U.S. patent application number 17/747646 was filed with the patent office on 2022-09-08 for dna vectors, transposons and transposases for eukaryotic genome modification.
The applicant listed for this patent is DNA TWOPOINTO INC.. Invention is credited to Kate Caves, Sridhar Govindrajan, Maggie Lee, Jeremy Minshull, Jon Ness, Mark Welch.
Application Number | 20220282260 17/747646 |
Document ID | / |
Family ID | 1000006348555 |
Filed Date | 2022-09-08 |
United States Patent
Application |
20220282260 |
Kind Code |
A1 |
Minshull; Jeremy ; et
al. |
September 8, 2022 |
DNA VECTORS, TRANSPOSONS AND TRANSPOSASES FOR EUKARYOTIC GENOME
MODIFICATION
Abstract
The present invention provides polynucleotide vectors for high
expression of heterologous genes. Some vectors further comprise
novel transposons and transposases that further improve expression.
Further disclosed are vectors that can be used in a gene transfer
system for stably introducing nucleic acids into the DNA of a cell.
The gene transfer systems can be used in methods, for example, gene
expression, bioprocessing, gene therapy, insertional mutagenesis,
or gene discovery.
Inventors: |
Minshull; Jeremy; (Los
Altos, CA) ; Welch; Mark; (Fremont, CA) ;
Govindrajan; Sridhar; (Los Altos, CA) ; Lee;
Maggie; (San Jose, CA) ; Caves; Kate; (San
Jose, CA) ; Ness; Jon; (Redwood City, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DNA TWOPOINTO INC. |
Newark |
CA |
US |
|
|
Family ID: |
1000006348555 |
Appl. No.: |
17/747646 |
Filed: |
May 18, 2022 |
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16726163 |
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17747646 |
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16140433 |
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62373422 |
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62239109 |
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61977474 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2840/203 20130101;
C12N 15/67 20130101; C12N 9/12 20130101; C12Y 207/07 20130101; C12N
1/185 20210501; C12N 2800/90 20130101; C12N 15/815 20130101; C12N
1/165 20210501; C12N 15/8509 20130101; C12R 2001/84 20210501; C07K
2319/09 20130101; C12N 15/1082 20130101; C12N 15/625 20130101; C12N
15/85 20130101; C12N 15/63 20130101; C12Y 207/00 20130101; C12N
2830/40 20130101; C12R 2001/865 20210501; C12N 2830/007 20130101;
C12N 15/52 20130101; C12N 9/1241 20130101; C12N 15/90 20130101;
C12N 15/81 20130101; C12N 2830/42 20130101 |
International
Class: |
C12N 15/67 20060101
C12N015/67; C12N 9/12 20060101 C12N009/12; C12N 15/90 20060101
C12N015/90; C12N 1/16 20060101 C12N001/16; C12N 1/18 20060101
C12N001/18; C12N 15/10 20060101 C12N015/10; C12N 15/52 20060101
C12N015/52; C12N 15/62 20060101 C12N015/62; C12N 15/63 20060101
C12N015/63; C12N 15/81 20060101 C12N015/81; C12N 15/85 20060101
C12N015/85 |
Claims
1-19. (canceled)
20. A method of creating a transgenic mammalian cell comprising:
introducing into a mammalian cell. a) a transposon comprising a
heterologous polynucleotide flanked by a pair of transposon ends,
wherein one transposon end comprises at least 16 contiguous
nucleotides from SEQ ID NO:25 and the other transposon end
comprises at least 16 contiguous nucleotides from SEQ ID NO:31, and
wherein the heterologous polynucleotide comprises a promoter that
is active in the mammalian cell; and b) a Bombyx transposase
comprising the amino acid sequence of SEQ ID NO:407; wherein the
transposase integrates the transposon into the genomic DNA of the
mammalian cell.
21. The method of claim 20, wherein one transposon end comprises a
sequence that is at least 90% identical to SEQ ID NO: 25 and the
other transposon end comprises a sequence that is at least 90%
identical to SEQ ID NO: 31.
22. The method of claim 20, wherein the heterologous polynucleotide
comprises a promoter selected from an EF1a promoter, a CMV
promoter, an EEF2 promoter, a GAPDH promoter, a Herpes Simplex
Virus thymidine kinase (HSV-TK) promoter, an actin promoter, a PGK
promoter, and an ubiquitin promoter.
23. The method of claim 20, wherein the heterologous polynucleotide
further comprises a second promoter, and wherein the transcription
directions from the first and second promoters are different.
24. The method of claim 20, wherein the promoter is operably linked
to one or more of: i) an open reading frame; ii) a selectable
marker; iii) a counter-selectable marker; iii) a nucleic acid
encoding a regulatory protein; iv) a nucleic acid encoding an
inhibitory RNA.
25. The method of claim 20, wherein the heterologous polynucleotide
comprises one or more sequence elements that increase expression by
enhancing RNA processing or export from the nucleus.
26. The method of claim 25, wherein the sequence elements are
selected from WPRE, HPRE (SEQ ID NOS:104-105), SAR (SEQ ID
NOS:108-111), AGS (SEQ ID NOS:106-107).
27. The method of claim 20, wherein the heterologous polynucleotide
comprises an insulator.
28. The method of claim 30, wherein the insulator comprises a
sequence that is at least 95% identical to a sequence selected from
SEQ ID NOS:859-865.
29. The method of claim 20, wherein the heterologous polynucleotide
comprises a gene encoding an antibody chain or gene encoding a
chimeric antigen receptor.
30. The method of claim 20, wherein the transposase further
comprises a heterologous nuclear localization signal (NLS) fused to
the transposase.
31. The method of claim 20, wherein the transposase is provided as
a protein.
32. The method of claim 20, wherein the transposase is provided as
a nucleic acid encoding the transposase.
33. The method of claim 32, wherein the nucleic acid is an mRNA
molecule.
34. The method of claim 33, wherein the mRNA molecule is expressed
from a promoter operably linked to a gene encoding the
transposase.
35. The method of claim 32, wherein the nucleic acid further
encodes a DNA binding domain (DBD) expressible as a fusion protein
with the transposase.
36. The method of claim 35, wherein the mammalian cell is a hamster
cell, human cell or lymphocyte.
37. The method of claim 20, further comprising identifying a cell
with a stably integrated transposon.
38. The method of claim 37, wherein the transposon of (a) comprises
a gene encoding a selectable marker, and the identifying comprises
growing the mammalian cell under conditions that provide a
selective advantage to cells comprising said selectable marker.
39. The method of claim 38, wherein the gene encoding the
selectable marker is operably linked to a promoter which is at
least 95% identical to a sequence selected from SEQ ID NOS:
937-948.
40. The method of claim 38, wherein the selectable marker is one of
the following: glutamine synthase, dihydrofolate reductase,
puromycin-N acetyl transferase, blasticidin-S deaminase, hygromycin
phosphotransferase, aminoglycoside phosphotransferase,
nourseothircin N-acetyl transferase, or a protein that binds to
zeocin.
41. The method of claim 38, wherein the selectable marker comprises
a gene encoding a fluorescent protein or a transmembrane
protein.
42. The method of claim 38, wherein the identifying comprises using
flow cytometry.
43. The method of claim 20, wherein the heterologous polynucleotide
encodes a polypeptide, which is expressed in the mammalian cell.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a non-provisional of 62/239,109 filed
Oct. 8, 2015, 62/325,872 filed Apr. 21, 2016 and 62/373,422 filed
Aug. 11, 2016, each incorporated by reference in its entirety for
all purposes. The present application is also related to 61/977,474
filed Apr. 9, 2014, 62/003,397 filed May 17, 2014, 62/046,875 filed
Sep. 5, 2014, 62/046,705, filed Sep. 5, 2014, 62/069,656 filed Oct.
28, 2014, 62/120,522 filed Feb. 25, 2015 PCT/US2015/025209, filed
Apr. 9, 2015, each incorporated by reference in its entirety for
all purposes.
REFERENCE TO A SEQUENCE LISTING
[0002] The application refers to sequences disclosed in a txt file
named 486542_SEQLST.TXT, of 4,308,152 bytes, created Oct. 6, 2016,
incorporated by reference.
2. BACKGROUND OF THE INVENTION
[0003] The efficiency with which a first polynucleotide can effect
the integration of heterologous DNA into the genome of a target
cell depends on the configuration of sequence elements within the
polynucleotide. The expression levels of genes encoded by the
integrated heterologous DNA also depend on the configuration of
sequence elements within the integrated heterologous DNA. The
efficiency of integration, the size of the heterologous DNA
sequence that can be integrated, the number of copies of the
heterologous DNA sequence that are integrated into each genome and
the type of genomic loci where integration occurs can often be
further improved by placing the heterologous DNA into a
transposon.
[0004] Transposons comprise two ends that are recognized by a
transposase. The transposase acts on the transposon to remove it
from one DNA molecule and integrate it into another. The DNA
between the two transposon ends is transposed by the transposase
along with the transposon ends. Heterologous DNA flanked by a pair
of transposon ends, such that it is recognized and transposed by a
transposase is referred to herein as a synthetic transposon.
Introduction of a synthetic transposon and a corresponding
transposase into the nucleus of a eukaryotic cell may result in
transposition of the transposon into the genome of the cell. More
active (hyperactive) transposons and transposases result in a
higher frequency of transposition, leading to a higher fraction of
cells whose genomes contain an integrated copy of the transposon
and/or cells whose genomes contain a larger number of integrated
copies of the transposon. These outcomes are useful because they
increase transformation efficiencies and because they can increase
expression levels from integrated heterologous DNA. There is thus a
need in the art for hyperactive transposases and transposons.
[0005] Transposition by a piggyBac-like transposase is perfectly
reversible. The transposon is initially integrated at an
integration target sequence in a recipient DNA molecule, during
which the target sequence becomes duplicated at each end of the
transposon inverted terminal repeats (ITRs). Subsequent
transposition removes the transposon and restores the recipient DNA
to its former sequence, with the target sequence duplication and
the transposon removed. However, this is not sufficient to remove a
transposon from a genome into which it has been integrated, as it
is highly likely that the transposon will be excised from the first
integration target sequence but integrated into a second
integration target sequence in the genome. Transposases that are
deficient for the integration function, on the other hand, can
excise the transposon from the first target sequence, but will be
unable to integrate into a second target sequence.
Integration-deficient transposases are thus useful for reversing
the genomic integration of a transposon.
3. SUMMARY OF THE INVENTION
[0006] Heterologous gene expression from polynucleotide constructs
that stably integrate into a target cell genome can be improved by
placing the expression polynucleotide between a pair of transposon
ends: sequence elements that are recognized and transposed by
transposases. DNA sequences inserted between a pair of transposon
ends can be excised by a transposase from one DNA molecule and
inserted into a second DNA molecule. Two novel piggyBac-like
transposon-transposase systems are disclosed that are not derived
from the looper moth Trichoplusia ni; one is derived from the
silkworm Bombyx mori and the other is derived from the frog Xenopus
tropicalis. Each of these comprises sequences that function as
transposon ends and that can be used in conjunction with a
corresponding transposase that recognizes and acts on those
transposon ends, as gene transfer systems for stably introducing
nucleic acids into the DNA of a cell. Hyperactive and
integration-deficient transposase variants are also disclosed.
[0007] Thus, the invention provides sequences of hyperactive
Xenopus transposases that are at least 90% identical to SEQ ID NO:
61, and positions and amino acid substitutions that can be
introduced either to enhance transposase activity, or to maintain
function of the transposase The invention also provides sequences
of transposon ends comprising at least 16 contiguous bases from SEQ
ID NO: 7 and at least 16 contiguous bases from SEQ ID NO: 16, and
inverted terminal repeats SEQ ID NO: 19. These sequences, when
placed on either side of a heterologous polynucleotide, create a
synthetic Xenopus transposon which can be excised from a
polynucleotide by Xenopus transposases. The synthetic transposon
may be integrated into a target genome by a Xenopus
transposase.
[0008] The invention provides sequences of hyperactive Bombyx
transposases that are at least 90% identical to SEQ ID NO: 415, and
positions and amino acid substitutions that can be introduced
either to enhance transposase activity, or to maintain function of
the transposase The invention also provides sequences of transposon
ends comprising at least 16 contiguous bases from SEQ ID NO: 25 and
at least 16 contiguous bases from SEQ ID NO: 31, and inverted
terminal repeats that are at least 87% identical to SEQ ID NO: 33.
These sequences, when placed on either side of a heterologous
polynucleotide, create a synthetic Bombyx transposon which can be
excised from a polynucleotide by Bombyx transposases. The synthetic
transposon may be integrated into a target genome by a Bombyx
transposase.
[0009] The invention provides methods for integrating a
heterologous polynucleotide into the genome of a target cell, by
introducing a Xenopus transposon and a Xenopus transposase, or a
Bombyx transposase and a Bombyx transposon, into a target cell. The
transposase may be introduced as protein, or as a polynucleotide
encoding the transposase and expressible in the target cell.
[0010] The invention also provides vector configurations, including
transposon configurations, that are particularly advantageous for
expression of genes in mammalian systems.
[0011] The transposons and transposases of the present invention
can be used in methods, for example, but not limited to,
heterologous gene expression, gene therapy, insertional
mutagenesis, or gene discovery.
4. BRIEF DESCRIPTION OF THE CONSTRUCT COMPOSITIONS AND EXPERIMENTAL
PROCEDURES
4.1 Construct Compositions
[0012] SEQ ID NO: 39 contains a weak promoter (the murine
phosphoglycerate kinase (PGK) promoter, SEQ ID NO: 937), operably
linked to a single open reading frame encoding DasherGFP
translationally coupled via a CHYSEL sequence to puromycin N-acetyl
transferase, followed by the polyadenylation signal from human beta
globin.
[0013] SEQ ID NO: 40 comprises a weak promoter (the murine
phosphoglycerate kinase (PGK) promoter, SEQ ID NO: 937), operably
linked to an open reading frame encoding puromycin N-acetyl
transferase, followed by the polyadenylation signal from human beta
globin. SEQ ID NO: 40 also comprises the EF1a promoter operably
linked to a gene encoding DasherGFP followed by expression
enhancing elements SEQ ID 866 and the rabbit globin polyadenylation
sequence.
4.2 Experimental Procedures
4.2.1 Transfection and Selection of CHO-K1
[0014] CHO-K1 cells (from ATCC) were grown in F12-K (from ATCC)+10%
FBS (from ATCC)+1% Penicillin-streptomycin (from ATCC) at
37.degree. C., 5% CO.sub.2 to 80% confluence. 500,000 cells were
plated in 24-well tissue culture plates and incubated at 37.degree.
C., 5% CO.sub.2 for 24 hours prior to transfection. Transfections
were performed in triplicate. Each transfection used a total of
500-1,000 ng DNA with Roche Extreme Gene 9 reagent (2:1 ratio) as
per manufacturer's protocol. Media with 50 .mu.g/ml puromycin was
added 72 hours post transfection. Puromycin selection was carried
out for 72 hours, after which puromycin was removed. Cells were
grown for 14 days post puromycin selection with two passages and
changes of media. Cells were harvested by scraping and measured in
a fluorimetric plate reader.
4.2.2 Transfection and Selection of CHO-S
[0015] CHO-S cells (from ATCC) were grown in CHOgro expression
medium (from Mirus) at 37.degree. C., 5% CO.sub.2 and seeded at
2.times.10.sup.6 cells/ml. 1 ml of cells were transfected with 1
.mu.g total nucleic acid. Transfections were performed in
duplicate. Each transfection used Mirus Transit-Pro and Mirus
TransIT-mRNA reagent as per manufacturer's protocol. Media with
puromycin was added 72 hours post transfection. Puromycin selection
was carried out for the number of days indicated, with a complete
media change into fresh puromycin-containing media after 5
days.
4.2.3 mRNA Preparation
[0016] mRNA encoding transposases was prepared by in vitro
transcription using T7 RNA polymerase. The mRNA comprised a 5'
sequence SEQ ID NO: 699 preceding the sequence encoding the open
reading frame, and a 3' sequence SEQ ID NO: 700 following the stop
codon at the end of the open reading frame. The mRNA had an
anti-reverse cap analog (3'-O-Me-m.sup.7G(5')ppp(5')G, and was
fully substituted with pseudo-uridine and 5-methyl-cytosine.
5. DETAILED DESCRIPTION OF THE INVENTION
5.1 Definitions
[0017] Use of the singular forms "a," "an," and "the" include
plural references unless the context clearly dictates otherwise.
Thus, for example, reference to "a polynucleotide" includes a
plurality of polynucleotides, reference to "a substrate" includes a
plurality of such substrates, reference to "a variant" includes a
plurality of variants, and the like.
[0018] Terms such as "connected," "attached," "linked," and
"conjugated" are used interchangeably herein and encompass direct
as well as indirect connection, attachment, linkage or conjugation
unless the context clearly dictates otherwise. Where a range of
values is recited, it is to be understood that each intervening
integer value, and each fraction thereof, between the recited upper
and lower limits of that range is also specifically disclosed,
along with each subrange between such values. The upper and lower
limits of any range can independently be included in or excluded
from the range, and each range where either, neither or both limits
are included is also encompassed within the invention. Where a
value being discussed has inherent limits, for example where a
component can be present at a concentration of from 0 to 100%, or
where the pH of an aqueous solution can range from 1 to 14, those
inherent limits are specifically disclosed. Where a value is
explicitly recited, it is to be understood that values which are
about the same quantity or amount as the recited value are also
within the scope of the invention. Where a combination is
disclosed, each sub combination of the elements of that combination
is also specifically disclosed and is within the scope of the
invention. Conversely, where different elements or groups of
elements are individually disclosed, combinations thereof are also
disclosed. Where any element of an invention is disclosed as having
a plurality of alternatives, examples of that invention in which
each alternative is excluded singly or in any combination with the
other alternatives are also hereby disclosed; more than one element
of an invention can have such exclusions, and all combinations of
elements having such exclusions are hereby disclosed.
[0019] Unless defined otherwise herein, all technical and
scientific terms used herein have the same meaning as commonly
understood by one of ordinary skill in the art to which this
invention belongs. Singleton, et al., Dictionary of Microbiology
and Molecular Biology, 2nd Ed., John Wiley and Sons, New York
(1994), and Hale & Marham, The Harper Collins Dictionary of
Biology, Harper Perennial, NY, 1991, provide one of skill with a
general dictionary of many of the terms used in this invention.
Although any methods and materials similar or equivalent to those
described herein can be used in the practice or testing of the
present invention, the preferred methods and materials are
described. Unless otherwise indicated, nucleic acids are written
left to right in 5' to 3' orientation; amino acid sequences are
written left to right in amino to carboxy orientation,
respectively. The terms defined immediately below are more fully
defined by reference to the specification as a whole.
[0020] The "configuration" of a polynucleotide means the functional
sequence elements within the polynucleotide, and the order and
direction of those elements.
[0021] The terms "corresponding transposon" and "corresponding
transposase" are used to indicate an activity relationship between
a transposase and a transposon. A transposase transposases its
corresponding transposon. Many transposases may correspond with a
single transposon, for example all of SEQ ID NOS: 52-402 are
corresponding transposases for transposon SEQ ID NO: 44). A
transposon is transposed by its corresponding transposase. Many
transposons may correspond with a single transposase, for example
the transposons shown in Table 5 rows 4-21 are all corresponding
transposons for transposase SEQ ID NO: 48.
[0022] The term "counter-selectable marker" means a polynucleotide
sequence that confers a selective disadvantage on a host cell.
Examples of counter-selectable markers include sacB, rpsL, tetAR,
pheS, thyA, gata-1, ccdB, kid and barnase (Bernard, 1995,
Journal/Gene, 162: 159-160; Bernard et al., 1994. Journal/Gene,
148: 71-74; Gabant et al., 1997, Journal/Biotechniques, 23:
938-941; Gababt et al., 1998, Journal/Gene, 207: 87-92; Gababt et
al., 2000, Journal/Biotechniques, 28: 784-788; Galvao and de
Lorenzo, 2005, Journal/Appl Environ Microbiol, 71: 883-892; Hartzog
et al., 2005, Joumal/Yeat, 22:789-798; Knipfer et al., 1997,
Journal/Plasmid, 37: 129-140; Reyrat et al., 1998, Journal/Infect
Immun, 66: 4011-4017; Soderholm et al., 2001,
Journal/Biotechniques, 31: 306-310, 312; Tamura et al., 2005,
Journal/Appl Environ Microbiol, 71: 587-590; Yazynin et al., 1999,
Journal/FEBS Lett, 452: 351-354). Counter-selectable markers often
confer their selective disadvantage in specific contexts. For
example, they may confer sensitivity to compounds that can be added
to the environment of the host cell, or they may kill a host with
one genotype but not kill a host with a different genotype.
Conditions which do not confer a selective disadvantage on a cell
carrying a counter-selectable marker are described as "permissive".
Conditions which do confer a selective disadvantage on a cell
carrying a counter-selectable marker are described as
"restrictive".
[0023] The term "coupling element" or "translational coupling
element" means a DNA sequence that allows the expression of a first
polypeptide to be linked to the expression of a second polypeptide.
Internal ribosome entry site elements (IRES elements) and
cis-acting hydrolase elements (CHYSEL elements) are examples of
coupling elements.
[0024] The terms "DNA sequence", "RNA sequence" or "polynucleotide
sequence" mean a contiguous nucleic acid sequence. The sequence can
be an oligonucleotide of 2 to 20 nucleotides in length to a full
length genomic sequence of thousands or hundreds of thousands of
base pairs.
[0025] The term "expression construct" means any polynucleotide
designed to transcribe an RNA. For example, a construct that
contains at least one promoter which is or may be operably linked
to a downstream gene, coding region, or polynucleotide sequence
(for example, a cDNA or genomic DNA fragment that encodes a
polypeptide or protein, or an RNA effector molecule, for example,
an antisense RNA, triplex-forming RNA, ribozyme, an artificially
selected high affinity RNA ligand (aptamer), a double-stranded RNA,
for example, an RNA molecule comprising a stem-loop or hairpin
dsRNA, or a bi-finger or multi-finger dsRNA or a microRNA, or any
RNA). An "expression vector" is a polynucleotide comprising a
promoter which can be operably linked to a second polynucleotide.
Transfection or transformation of the expression construct into a
recipient cell allows the cell to express an RNA effector molecule,
polypeptide, or protein encoded by the expression construct. An
expression construct may be a genetically engineered plasmid,
virus, recombinant virus, or an artificial chromosome derived from,
for example, a bacteriophage, adenovirus, adeno-associated virus,
retrovirus, lentivirus, poxvirus, or herpesvirus. Such expression
vectors can include sequences from bacteria, viruses or phages.
Such vectors include chromosomal, episomal and virus-derived
vectors, for example, vectors derived from bacterial plasmids,
bacteriophages, yeast episomes, yeast chromosomal elements, and
viruses, vectors derived from combinations thereof, such as those
derived from plasmid and bacteriophage genetic elements, cosmids
and phagemids. An expression construct can be replicated in a
living cell, or it can be made synthetically. For purposes of this
application, the terms "expression construct", "expression vector",
"vector", and "plasmid" are used interchangeably to demonstrate the
application of the invention in a general, illustrative sense, and
are not intended to limit the invention to a particular type of
expression construct.
[0026] The term "expression polypeptide" means a polypeptide
encoded by a gene on an expression construct.
[0027] The term "expression system" means any in vivo or in vitro
biological system that is used to produce one or more gene product
encoded by a polynucleotide.
[0028] A "gene transfer system" comprises a vector or gene transfer
vector, or a polynucleotide comprising the gene to be transferred
which is cloned into a vector (a "gene transfer polynucleotide" or
"gene transfer construct"). A gene transfer system may also
comprise other features to facilitate the process of gene transfer.
For example, a gene transfer system may comprise a vector and a
lipid or viral packaging mix for enabling a first polynucleotide to
enter a cell, or it may comprise a polynucleotide that includes a
transposon and a second polynucleotide sequence encoding a
corresponding transposase to enhance productive genomic integration
of the transposon. The transposases and transposons of a gene
transfer system may be on the same nucleic acid molecule or on
different nucleic acid molecules. The transposase of a gene
transfer system may be provided as a polynucleotide or as a
polypeptide.
[0029] Two elements are "heterologous" to one another if not
naturally associated. For example, a nucleic acid sequence encoding
a protein linked to a heterologous promoter means a promoter other
than that which naturally drives expression of the protein. A
heterologous nucleic acid flanked by transposon ends or ITRs means
a heterologous nucleic acid not naturally flanked by those
transposon ends or ITRs, such as a nucleic acid encoding a
polypeptide other than a transposase, including an antibody heavy
or light chain. A nucleic acid is heterologous to a cell if not
naturally found in the cell or if naturally found in the cell but
in a different location (e.g., episomal or different genomic
location) than the location described.
[0030] The term "host" means any prokaryotic or eukaryotic organism
that can be a recipient of a nucleic acid. A "host," as the term is
used herein, includes prokaryotic or eukaryotic organisms that can
be genetically engineered. For examples of such hosts, see Maniatis
et al., Molecular Cloning. A Laboratory Manual, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y. (1982). As used herein, the
terms "host," "host cell," "host system" and "expression host" can
be used interchangeably.
[0031] A "hyperactive" transposase is a transposase that is more
active than the naturally occurring transposase from which it is
derived. "Hyperactive" transposases are thus not naturally
occurring sequences. Hyperactive Xenopus transposases are those
that are more active than SEQ ID NO: 48. Hyperactive Bombyx
transposases are those that are more active than SEQ ID NO:
407.
[0032] `Integration defective` means a transposase that can excise
its corresponding transposon, but that integrates the excised
transposon at a lower frequency into the host genome than a
corresponding naturally occurring transposase. Integration
defective Xenopus transposases are deficient relative to SEQ ID NO:
48. Integration defective Bombyx transposases are deficient
relative to SEQ ID NO: 407.
[0033] An "IRES" or "internal ribosome entry site" means a
specialized sequence that directly promotes ribosome binding,
independent of a cap structure.
[0034] An `isolated` polypeptide or polynucleotide means a
polypeptide or polynucleotide that has been either removed from its
natural environment, produced using recombinant techniques, or
chemically or enzymatically synthesized. Polypeptides or
polynucleotides of this invention may be purified, that is,
essentially free from any other polypeptide or polynucleotide and
associated cellular products or other impurities.
[0035] The terms "nucleoside" and "nucleotide" include those
moieties which contain not only the known purine and pyrimidine
bases, but also other heterocyclic bases which have been modified.
Such modifications include methylated purines or pyrimidines,
acylated purines or pyrimidines, or other heterocycles. Modified
nucleosides or nucleotides can also include modifications on the
sugar moiety, for example, where one or more of the hydroxyl groups
are replaced with halogen, aliphatic groups, or is functionalized
as ethers, amines, or the like. The term "nucleotidic unit" is
intended to encompass nucleosides and nucleotides.
[0036] An "Open Reading Frame" or "ORF" means a portion of a
polynucleotide that, when translated into amino acids, contains no
stop codons. The genetic code reads DNA sequences in groups of
three base pairs, which means that a double-stranded DNA molecule
can read in any of six possible reading frames-three in the forward
direction and three in the reverse. An ORF typically also includes
an initiation codon at which translation may start.
[0037] The term "operably linked" refers to functional linkage
between two sequences such that one sequence modifies the behavior
of the other. For example, a first polynucleotide comprising a
nucleic acid expression control sequence (such as a promoter, IRES
sequence, enhancer or array of transcription factor binding sites)
and a second polynucleotide are operably linked if the first
polynucleotide affects transcription and/or translation of the
second polynucleotide. Similarly, a first amino acid sequence
comprising a secretion signal or a subcellular localization signal
and a second amino acid sequence are operably linked if the first
amino acid sequence causes the second amino acid sequence to be
secreted or localized to a subcellular location.
[0038] The term "overhang" or "DNA overhang" means the
single-stranded portion at the end of a double-stranded DNA
molecule. Complementary overhangs are those which will base-pair
with each other.
[0039] A "piggyBac-like transposase" means a transposase with at
least 20% sequence identity as identified using the TBLASTN
algorithm to the piggyBac transposase from Trichoplusia ni (SEQ ID
NO: 698), and as more fully described in Sakar, A. et. al., (2003).
Mol. Gen. Genomics 270: 173-180. "Molecular evolutionary analysis
of the widespread piggyBac transposon family and related
`domesticated` species", and further characterized by a DDE-like
DDD motif, with aspartate residues at positions corresponding to
D268, D346, and D447 of Trichoplusia ni piggyBac transposase on
maximal alignment. PiggyBac-like transposases are also
characterized by their ability to excise their transposons
precisely with a high frequency. A "piggyBac-like transposon" means
a transposon having transposon ends which are the same or at least
80% and preferably at least 90, 95, 96, 97, 98 or 99% identical to
the transposon ends of a naturally occurring transposon that
encodes a piggyBac-like transposase. A piggyBac-like transposon
includes an inverted terminal repeat (ITR) sequence of
approximately 12-16 bases at each end, and is flanked on each side
by a 4 base sequence corresponding to the integration target
sequence which is duplicated on transposon integration (the Target
Site Duplication or Target Sequence Duplication or TSD).
PiggyBac-like transposons and transposases occur naturally in a
wide range of organisms including Argyrogramma agnate (GU477713),
Anopheles gambiae (XP_312615; XP_320414; XP_310729), Aphis gossypii
(GU329918), Acyrthosiphon pisum (XP_001948139), Agrotis ypsilon
(GU477714), Bombyx mori (BAD11135), Ciona intestinalis
(XP_002123602), Chilo suppressalis (JX294476), Drosophila
melanogaster (AAL39784), Daphnia pulicaria (AAM76342), Helicoverpa
armigera (ABS18391), Homo sapiens (NP_689808), Heliothis virescens
(ABD76335), Macdunnoughia crassisigna (EU287451), Macaca
fascicularis (AB179012), Mus musculus (NP_741958), Pectinophora
gossypiella (GU270322), Rattus norvegicus (XP 220453), Tribolium
castaneum (XP 001814566), Trichoplusia ni (AAA87375) and Xenopus
tropicalis (BAF82026), although transposition activity has been
described for almost none of these.
[0040] The terms "polynucleotide," "oligonucleotide," "nucleic
acid" and "nucleic acid molecule" and "gene" are used
interchangeably to refer to a polymeric form of nucleotides of any
length, and may comprise ribonucleotides, deoxyribonucleotides,
analogs thereof, or mixtures thereof. This term refers only to the
primary structure of the molecule. Thus, the term includes triple-,
double- and single-stranded deoxyribonucleic acid ("DNA"), as well
as triple-, double- and single-stranded ribonucleic acid ("RNA").
It also includes modified, for example by alkylation, and/or by
capping, and unmodified forms of the polynucleotide. More
particularly, the terms "polynucleotide," "oligonucleotide,"
"nucleic acid" and "nucleic acid molecule" include
polydeoxyribonucleotides (containing 2-deoxy-D-ribose),
polyribonucleotides (containing D-ribose), including tRNA, rRNA,
hRNA, siRNA and mRNA, whether spliced or unspliced, any other type
of polynucleotide which is an N- or C-glycoside of a purine or
pyrimidine base, and other polymers containing nonnucleotidic
backbones, for example, polyamide (for example, peptide nucleic
acids ("PNAs")) and polymorpholino (commercially available from the
Anti-Virals, Inc., Corvallis, Oreg., as Neugene) polymers, and
other synthetic sequence-specific nucleic acid polymers providing
that the polymers contain nucleobases in a configuration which
allows for base pairing and base stacking, such as is found in DNA
and RNA. There is no intended distinction in length between the
terms "polynucleotide," "oligonucleotide," "nucleic acid" and
"nucleic acid molecule," and these terms are used interchangeably
herein. These terms refer only to the primary structure of the
molecule. Thus, these terms include, for example, 3'-deoxy-2',
5'-DNA, oligodeoxyribonucleotide N3' P5' phosphoramidates,
2'-O-alkyl-substituted RNA, double- and single-stranded DNA, as
well as double- and single-stranded RNA, and hybrids thereof
including for example hybrids between DNA and RNA or between PNAs
and DNA or RNA, and also include known types of modifications, for
example, labels, alkylation, "caps," substitution of one or more of
the nucleotides with an analog, internucleotide modifications such
as, for example, those with uncharged linkages (for example, methyl
phosphonates, phosphotriesters, phosphoramidates, carbamates, or
the like) with negatively charged linkages (for example,
phosphorothioates, phosphorodithioates, or the like), and with
positively charged linkages (for example,
aminoalkylphosphoramidates, aminoalkylphosphotriesters), those
containing pendant moieties, such as, for example, proteins
(including enzymes (for example, nucleases), toxins, antibodies,
signal peptides, poly-L-lysine, or the like), those with
intercalators (for example, acridine, psoralen, or the like), those
containing chelates (of, for example, metals, radioactive metals,
boron, oxidative metals, or the like), those containing alkylators,
those with modified linkages (for example, alpha anomeric nucleic
acids, or the like), as well as unmodified forms of the
polynucleotide or oligonucleotide.
[0041] A "promoter" means a nucleic acid sequence sufficient to
direct transcription of an operably linked nucleic acid molecule.
Also included in this definition are those transcription control
elements (for example, enhancers) that are sufficient to render
promoter-dependent gene expression controllable in a cell
type-specific, tissue-specific, or temporal-specific manner, or
that are inducible by external signals or agents; such elements,
may be within the 3' region of a gene or within an intron.
Desirably, a promoter is operably linked to a nucleic acid
sequence, for example, a cDNA or a gene sequence, or an effector
RNA coding sequence, in such a way as to enable expression of the
nucleic acid sequence, or a promoter is provided in an expression
cassette into which a selected nucleic acid sequence to be
transcribed can be conveniently inserted.
[0042] The term "selectable marker" means a polynucleotide segment
that allows one to select for or against a molecule or a cell that
contains it, often under particular conditions. These markers can
encode an activity, such as, but not limited to, production of RNA,
peptide, or protein, or can provide a binding site for RNA,
peptides, proteins, inorganic and organic compounds or
compositions. Examples of selectable markers include but are not
limited to: (1) DNA segments that encode products which provide
resistance against otherwise toxic compounds (e.g., antibiotics);
(2) DNA segments that encode products which are otherwise lacking
in the recipient cell (e.g., tRNA genes, auxotrophic markers); (3)
DNA segments that encode products which suppress the activity of a
gene product; (4) DNA segments that encode products which can be
readily identified (e.g., phenotypic markers such as
beta-galactosidase, green fluorescent protein (GFP), and cell
surface proteins); (5) DNA segments that bind products which are
otherwise detrimental to cell survival and/or function; (6) DNA
segments that otherwise inhibit the activity of any of the DNA
segments described in Nos. 1-5 above (e.g., antisense
oligonucleotides); (7) DNA segments that bind products that modify
a substrate (e.g. restriction endonucleases); (8) DNA segments that
can be used to isolate a desired molecule (e.g. specific protein
binding sites); (9) DNA segments that encode a specific nucleotide
sequence which can be otherwise non-functional (e.g., for PCR
amplification of subpopulations of molecules); and/or (10) DNA
segments, which when absent, directly or indirectly confer
sensitivity to particular compounds.
[0043] Sequence identity can be determined by aligning sequences
using algorithms, such as BESTFIT, FASTA, and TFASTA in the
Wisconsin Genetics Software Package Release 7.0, Genetics Computer
Group, 575 Science Dr., Madison, Wis.), using default gap
parameters, or by inspection, and the best alignment (i.e.,
resulting in the highest percentage of sequence similarity over a
comparison window). Percentage of sequence identity is calculated
by comparing two optimally aligned sequences over a window of
comparison, determining the number of positions at which the
identical residues occurs in both sequences to yield the number of
matched positions, dividing the number of matched positions by the
total number of matched and mismatched positions not counting gaps
in the window of comparison (i.e., the window size), and
multiplying the result by 100 to yield the percentage of sequence
identity. Unless otherwise indicated the window of comparison
between two sequences is defined by the entire length of the
shorter of the two sequences.
[0044] A "target nucleic acid" is a nucleic acid into which a
transposon is to be inserted. Such a target can be part of a
chromosome, episome or vector.
[0045] An "integration target sequence" or "target sequence" or
"target site" for a transposase is a site or sequence in a target
DNA molecule into which a transposon can be inserted by a
transposase. The piggyBac transposase from Trichoplusia ni inserts
its transposon predominantly into the target sequence 5'-TTAA-3'.
PiggyBac-like transposases transpose their transposons using a
cut-and-paste mechanism, which results in duplication of their 4
base pair target sequence on insertion into a DNA molecule. The
target sequence is thus found on each side of an integrated
piggyBac-like transposon.
[0046] The term "translation" refers to the process by which a
polypeptide is synthesized by a ribosome `reading` the sequence of
a polynucleotide.
[0047] A `transposase` is a polypeptide that catalyzes the excision
of a corresponding transposon from a donor polynucleotide, for
example a vector, and (providing the transposase is not
integration-deficient) the subsequent integration of the transposon
into a target nucleic acid. A "Bombyx transposase" means a
transposase with at least 80% sequence identity to SEQ ID NO 407,
including hyperactive variants of SEQ ID NO 407, that are able to
transposase a corresponding transposon. A "Xenopus transposase"
means a transposase with at least 80% sequence identity to SEQ ID
NO 48, including hyperactive variants of SEQ ID NO 48, that are
able, when fused to a heterologous nuclear localization sequence,
to transposase a corresponding transposon.
[0048] The term "transposition" is used herein to mean the action
of a transposase in excising a transposon from one polynucleotide
and then integrating it, either into a different site in the same
polynucleotide, or into a second polynucleotide.
[0049] The term "transposon" means a polynucleotide that can be
excised from a first polynucleotide, for instance, a vector, and be
integrated into a second position in the same polynucleotide, or
into a second polynucleotide, for instance, the genomic or
extrachromosomal DNA of a cell, by the action of a corresponding
trans-acting transposase. A transposon comprises a first transposon
end and a second transposon end, which are polynucleotide sequences
recognized by and transposed by a transposase. A transposon usually
further comprises a first polynucleotide sequence between the two
transposon ends, such that the first polynucleotide sequence is
transposed along with the two transposon ends by the action of the
transposase. Natural transposons frequently comprise DNA encoding a
transposase that acts on the transposon. Transposons of the present
invention are "synthetic transposons" comprising a heterologous
polynucleotide sequence which is transposable by virtue of its
juxtaposition between two transposon ends.
[0050] The term "transposon end" means the cis-acting nucleotide
sequences that are sufficient for recognition by and transposition
by a corresponding transposase. Transposon ends of piggyBac-like
transposons comprise perfect or imperfect repeats such that the
respective repeats in the two transposon ends are reverse
complements of each other. These are referred to as inverted
terminal repeats (ITR) or terminal inverted repeats (TIR). A
transposon end may or may not include additional sequence proximal
to the ITR that promotes or augments transposition.
[0051] The term "vector" or "DNA vector" or "gene transfer vector"
refers to a polynucleotide that is used to perform a "carrying"
function for another polynucleotide. For example, vectors are often
used to allow a polynucleotide to be propagated within a living
cell, or to allow a polynucleotide to be packaged for delivery into
a cell, or to allow a polynucleotide to be integrated into the
genomic DNA of a cell. A vector may further comprise additional
functional elements, for example it may comprise a transposon.
5.2 Description
5.2.1 Genomic Integration
[0052] Expression of a gene from a heterologous polynucleotide in a
eukaryotic host cell can be improved if the heterologous
polynucleotide is integrated into the genome of the host cell.
Integration of a polynucleotide into the genome of a host cell also
generally makes it stably heritable, by subjecting it to the same
mechanisms that ensure the replication and division of genomic DNA.
Such stable heritability is desirable for achieving good and
consistent expression over long growth periods. For manufacturing
of biomolecules, particularly for therapeutic applications, the
stability of the host and consistency of expression levels is also
important for regulatory purposes. Cells with gene transfer
vectors, including transposon-based gene transfer vectors,
integrated into their genomes are thus an important aspect of the
invention.
[0053] Heterologous polynucleotides may be more efficiently
integrated into a target genome if they are part of a transposon,
for example so that they may be integrated by a transposase. A
particular benefit of a transposon is that the entire
polynucleotide between the transposon ITRs is integrated. This is
in contrast to random integration, where a polynucleotide
introduced into a eukaryotic cell is often fragmented at random in
the cell, and only parts of the polynucleotide become incorporated
into the target genome, usually at a low frequency. The piggyBac
transposon from the looper moth Trichoplusia ni has been shown to
be transposed by its transposase in cells from many organisms (see
e.g. Keith et al (2008) BMC Molecular Biology 9:72 "Analysis of the
piggyBac transposase reveals a functional nuclear targeting signal
in the 94 c-terminal residues"). Heterologous polynucleotides
incorporated into piggyBac-like transposons may be integrated into
eukaryotic cells including animal cells, fungal cells or plant
cells. Preferred animal cells can be vertebrate or invertebrate.
Preferred vertebrate cells include cells from mammals including
rodents such as rats, mice, and hamsters; ungulates, such as cows,
goats or sheep; and swine. Preferred vertebrate cells also include
cells from human tissues and human stem cells. Target cells types
include lymphocytes, hepatocytes, neural cells, muscle cells, blood
cells, embryonic stem cells, somatic stem cells, hematopoietic
cells, embryos, zygotes and sperm cells (some of which are open to
be manipulated in an in vitro setting). Preferred cells can be
pluripotent cells (cells whose descendants can differentiate into
several restricted cell types, such as hematopoietic stem cells or
other stem cells) or totipotent cells (i.e., a cell whose
descendants can become any cell type in an organism, e.g.,
embryonic stem cells). Preferred culture cells are Chinese hamster
ovary (CHO) cells or Human embryonic kidney (HEK293) cells.
Preferred fungal cells are yeast cells including Saccharomyces
cerevisiae and Pichia pastoris. Preferred plant cells are algae,
for example Chlorella, tobacco, maize and rice (Nishizawa-Yokoi et
al (2014) Plant J. 77:454-63 "Precise marker excision system using
an animal derived piggyBac transposon in plants").
[0054] Preferred gene transfer systems comprise a transposon in
combination with a corresponding transposase protein that
transposases the transposon, or a nucleic acid that encodes the
corresponding transposase protein and is expressible in the target
cell.
[0055] A transposase protein can be introduced into a cell as a
protein or as a nucleic acid encoding the transposase, for example
as a ribonucleic acid, including mRNA or any polynucleotide
recognized by the translational machinery of a cell; as DNA, e.g.
as extrachromosomal DNA including episomal DNA; as plasmid DNA, or
as viral nucleic acid. Furthermore, the nucleic acid encoding the
transposase protein can be transfected into a cell as a nucleic
acid vector such as a plasmid, or as a gene expression vector,
including a viral vector. The nucleic acid can be circular or
linear. DNA encoding the transposase protein can be stably inserted
into the genome of the cell or into a vector for constitutive or
inducible expression. Where the transposase protein is transfected
into the cell or inserted into the vector as DNA, the transposase
encoding sequence is preferably operably linked to a heterologous
promoter. There are a variety of promoters that could be used
including constitutive promoters, tissue-specific promoters,
inducible promoters, and the like. All DNA or RNA sequences
encoding Bombyx or Xenopus transposase proteins are expressly
contemplated. Alternatively, the transposase may be introduced into
the cell directly as protein, for example using cell-penetrating
peptides (e.g. as described in Ramsey and Flynn (2015) Pharmacol.
Ther. 154: 78-86 "Cell-penetrating peptides transport therapeutics
into cells"); using small molecules including salt plus
propanebetaine (e.g. as described in Astolfo et al (2015) Cell 161:
674-690); or electroporation (e.g. as described in Morgan and Day
(1995) Methods in Molecular Biology 48: 63-71 "The introduction of
proteins into mammalian cells by electroporation").
[0056] It is possible to insert the transposon into DNA of a cell
through non-homologous recombination through a variety of
reproducible mechanisms, and even without the activity of a
transposase. The transposons described herein can be used for gene
transfer regardless of the mechanisms by which the genes are
transferred.
5.2.2 Xenopus-Derived Piggybac-Like Transposons
[0057] Natural DNA transposons undergo a `cut and paste` system of
replication in which the transposon is excised from a first DNA
molecule and inserted into a second DNA molecule. DNA transposons
are characterized by inverted terminal repeats (ITRs) and are
mobilized by an element-encoded transposase. The piggyBac
transposon/transposase system is particularly useful because of the
precision with which the transposon is integrated and excised (see
for example "Fraser, M. J. (2001) The TTAA-Specific Family of
Transposable Elements: Identification, Functional Characterization,
and Utility for Transformation of Insects. Insect Transgenesis:
Methods and Applications. A. M. Handler and A. A. James. Boca
Raton, Fla., CRC Press: 249-268"; and "US 20070204356 A1: PiggyBac
constructs in vertebrates" and references therein).
[0058] Many sequences with sequence similarity to the piggyBac
transposase from Trichoplusia ni have been found in the genomes of
phylogenetically distinct species from fungi to mammals, but very
few have been shown to possess transposase activity (see for
example Wu M, et al (2011) Genetica 139:149-54. "Cloning and
characterization of piggyBac-like elements in lepidopteran
insects", and references therein).
[0059] Excision activity has been identified in Txb transposases
from Xenopus (Hikosaka et. al., Mol. Biol. Evol., 24(12):2648-2656,
2007), but the authors reported no evidence for the integration of
the excised target into the genome. This report suggested such
transposases lack integration activity. However, surprisingly we
have found that transposases originally identified in the genome of
Xenopus tropicalis (SEQ ID NOS 48 and 49) are transpositionally
active in mammalian cells when fused to a heterologous nuclear
localization signal. In the absence of a fused nuclear localization
signal, the naturally occurring Xenopus transposases are
essentially inactive for genomic integration (see Example 6.1.1 and
Table 1). Our discovery reveals why Hirosaka failed to see
integration: the experiments performed by Hikosaka et. al. involved
transfecting a DNA target and DNA encoding a transposase into
mammalian cells. The transposase, produced in the cytoplasm, would
be able to act on transfected DNA in the cytoplasm to excise the
transposon. However, no genomic integration activity would be
detected if the transposase, which lacked an NLS, remained
cytoplasmic.
[0060] Here we have identified transposon ends including ITRs that,
when added to the ends of a heterologous polynucleotide sequence,
create a synthetic Xenopus transposon which is efficiently
integrated into genomic DNA by a Xenopus transposase. A left target
sequence followed by a left transposon end sequence comprising a
sequence selected from SEQ ID NO: 1-8 is added to on one side of a
heterologous polynucleotide. A right transposon end sequence
comprising a sequence selected from SEQ ID NO: 12-16, and followed
by a right target sequence is added to the other side of the
heterologous polynucleotide. The resulting polynucleotide is a
synthetic Xenopus transposon, and is efficiently transposed by
transposases selected from SEQ ID NO: 48 or 49, fused to a
heterologous nuclear localization signal. See Tables 1-3 and
Examples 6.1.1, 6.1.2.1 and 6.1.2.2.
[0061] Xenopus transposases recognize synthetic Xenopus
transposons. They excise the transposon from a first DNA molecule,
by cutting the DNA at the target sequence at the left end of one
transposon end and the target sequence at the right end of the
second transposon end, re-join the cut ends of the first DNA
molecule to leave a single copy of the target sequence. The excised
transposon sequence, including any heterologous DNA that is between
the transposon ends, is integrated by the transposase into a target
sequence of a second DNA molecule, such as the genome of a target
cell.
[0062] These Xenopus left and right transposon ends share a 14 bp
almost perfectly repeated sequence inverted in orientation in the
two ends: (5'-CCYTTTBMCTGCCA: SEQ ID NO: 19) adjacent to the target
sequence. Here and elsewhere when inverted repeats are defined by a
sequence including a nucleotide defined by an ambiguity code, the
identity of that nucleotide can be selected independently in the
two repeats. The near-perfect conservation of this 14 bp ITR
sequence at both ends of the Xenopus transposon allow us to
identify it as the transposon ITR. Transposons comprising a
heterologous polynucleotide inserted between two transposon ends,
each comprising SEQ ID NO: 19 in inverted orientations in the two
transposon ends, and flanked by a target sequence, can be
transposed from one DNA molecule to another, by their corresponding
Xenopus transposases. Naturally occurring Xenopus transposases (SEQ
ID NO: 48 and 49) must be fused to a heterologous nuclear
localization signal to effect this transposition.
[0063] Truncated and modified versions of naturally occurring left
and right transposon ends will function as part of a synthetic
Xenopus transposons. For example, as shown in Example 6.1.2.2 and
Tables 2 and 3, a left transposon end consisting of a target
sequence followed by a sequence selected from SEQ ID NO: 4-7, and a
right transposon end consisting of a sequence selected from SEQ ID
NO: 13-16 followed by a target sequence contains all sequences
necessary for transposition of DNA by a Xenopus transposase fused
to a heterologous nuclear localization signal. We observed that
sequence differences are tolerated within the truncated transposon
ends in addition to the degeneracies noted in the ITR sequences.
For example, left transposon end SEQ ID NO: 7 consists of SEQ ID
NO: 9 in addition to the ITR, while left transposon end SEQ ID NO:
5 consists of SEQ ID NO: 10 in addition to the ITR. Similarly,
right transposon end SEQ ID NO: 16 consists of SEQ ID NO: 17 in
addition to the ITR, while right transposon end SEQ ID NO: 13
consists of SEQ ID NO: 18 in addition to the ITR.
[0064] A Xenopus transposon can comprise a heterologous
polynucleotide flanked by two transposon ends, wherein one
transposon end comprises a sequence that is at least 90% identical
or at least 95% identical or at least 99% identical to SEQ ID NO: 7
and one transposon end comprises a sequence that is at least 90%
identical or at least 95% identical or at least 99% identical to
SEQ ID NO: 16.
[0065] A Xenopus transposon can comprise a heterologous
polynucleotide flanked by two transposon ends, wherein one
transposon end comprises at least 14 or at least 16 or at least 18
or at least 20 or at least 25 contiguous bases from SEQ ID NO: 7
and one transposon end comprises at least 14, or at least 16, or at
least 18, or at least 20 contiguous bases from SEQ ID NO: 16.
[0066] A Xenopus transposon can comprise a heterologous
polynucleotide flanked by two transposon ends wherein each
transposon end comprises the sequence 5'-CCYTTTBMCTGCCA-3' (SEQ ID
NO: 19) inverted in orientation in the two transposon ends. One end
of this Xenopus transposon may further comprise at least 14, or at
least 16, or at least 18, or at least 20 contiguous bases from SEQ
ID NO: 9 and the other end may further comprise at least 14 or at
least 16 or at least 18 or at least 20 or at least 25 contiguous
bases from SEQ ID NO: 17.
[0067] Xenopus transposons are transposable by Xenopus
transposases, for example by at least one polypeptide selected from
SEQ ID NO: 48, 49 or 52-402 and fused to a heterologous nuclear
localization signal. Operability of a Xenopus transposon can be
shown by the ability of a transposase having the amino acid
sequence of SEQ ID NO:61 fused to a heterologous NLS to transpose
the transposon.
[0068] Cells whose genomes contain a Xenopus transposon are an
aspect of the invention. The cell may be any eukaryotic cell.
5.2.3 Bombyx-Derived Piggybac-Like Transposons
[0069] A transposon was identified from the genome of Bombyx mori
with the functional transposon ends being contained within SEQ ID
NO: 23 and SEQ ID NO: 29. A transposase that can recognize and
transpose a transposon comprising these transposon ends is SEQ ID
NO: 407. The inverted terminal repeats (ITRs) at the ends of the
natural transposon comprising SEQ ID NOS: 23 and 29 were not
flanked by the canonical 5'-TTAA-3' target sequence usually
observed for transposons with significant sequence identity to
Trichoplusia ni piggyBac; they were flanked by 5'-TTAT-3' sequences
adjacent to the ITRs.
[0070] Here we have identified transposon ends including ITRs that
can be added to the ends of a heterologous polynucleotide sequence
to effect the efficient integration of the polynucleotide into
genomic DNA by the action of a Bombyx transposase. A left target
sequence followed by a left transposon end sequence comprising a
sequence selected from SEQ ID NO: 23-27 is added to on one side of
a heterologous polynucleotide. A right transposon end sequence
comprising a sequence selected from SEQ ID NO: 29-32, followed by a
right target sequence is added to the other side of the
heterologous polynucleotide. The resulting polynucleotide is a
synthetic Bombyx transposon, and is efficiently transposed by
transposase SEQ ID NO: 407, whether or not fused to a heterologous
nuclear localization signal. See Tables 1 and 2 and Examples 6.1.1
and 6.1.2.1.
[0071] Bombyx transposases recognize synthetic Bombyx transposons.
They excise the transposon from a first DNA molecule, by cutting
the DNA at the target sequence at the left end of one transposon
end and the target sequence at the right end of the second
transposon end, re-join the cut ends of the first DNA molecule to
leave a single copy of the target sequence. The excised transposon
sequence, including any heterologous DNA that is between the
transposon ends, is integrated into a target sequence of a second
DNA molecule, such as the genome of a target cell.
[0072] The left and right Bombyx transposon ends share a 16 bp
repeat sequence at their ends (5'-CCCGGCGAGCATGAGG-3': SEQ ID NO:
33) inverted in orientation in the two ends immediately adjacent to
the target sequence. That is the left transposon end begins with
the sequence 5'-CCCGGCGAGCATGAGG-3' (SEQ ID NO: 33), and the right
transposon ends with the reverse complement of this sequence:
5'-CCTCATGCTCGCCGGG-3' (SEQ ID NO: 34). The perfect conservation of
this 16 bp sequence at both ends of the transposon allowed us to
identify it as the transposon ITR.
[0073] The degeneracy observed for the Xenopus piggyBac-like
transposon described in Section 5.2.2 suggests that this sequence
is not completely immutable, but may accept one or two or three
nucleotide changes from the consensus (as described for SEQ ID NO:
19), providing functional Bombyx ITRs with 93%, 87% or 81% sequence
identity with SEQ ID NO: 33 (or (SEQ ID NO: 34) respectively. A
Bombyx transposon can comprise a heterologous polynucleotide
inserted between a left and right transposon end, wherein each
transposon end comprises a sequence at least 81% identical or at
least 87% identical or at least 93% identical to the sequence
5'-CCCGGCGAGCATGAGG-3' (SEQ ID NO: 33) at one end, a sequence at
least 81% identical or at least 87% identical or at least 93%
identical to the sequence 5'-CCTCATGCTCGCCGGG-3' (SEQ ID NO: 34) at
the other end.
[0074] Truncated and modified versions of the left and right
transposon ends also function as part of a synthetic Bombyx
transposon. For example, as shown in Example 6.1.2.1 and Table 2, a
target sequence followed by a left transposon end comprising a
sequence selected from SEQ ID NO: 23-25, and a right transposon end
comprising SEQ ID NO: 29 or 31, followed by a target sequence,
contains all sequences necessary for transposition of by a Bombyx
transposase.
[0075] A Bombyx transposon can comprise a heterologous
polynucleotide flanked by two transposon ends wherein one
transposon end comprises a sequence that is at least 90% or at
least 95% identical or at least 99% identical to SEQ ID NO: 25 and
one transposon end comprises a sequence that is at least 90%
identical or at least 95% or at least 99% identical to SEQ ID NO:
31.
[0076] A Bombyx transposon can comprise a heterologous
polynucleotide flanked by two transposon ends, wherein one
transposon end comprises at least 14 or at least 16 or at least 18
or at least 20 contiguous bases from SEQ ID NO: 25 and one
transposon end comprises at least 14 or at least 16 or at least 18
or at least 20 contiguous bases from SEQ ID NO: 31.
[0077] A Bombyx transposon can comprise a heterologous
polynucleotide flanked by two transposon ends wherein each
transposon end comprises a sequence that is at least 81% identical
or at least 87% identical or at least 93% identical to the sequence
5'-CCCGGCGAGCATGAGG-3' (SEQ ID NO: 33) inverted in orientation in
the two transposon ends. One end of this Bombyx transposon may
further comprise at least 14, or at least 16, or at least 18, or at
least 20 contiguous bases from SEQ ID NO: 27 and the other end may
further comprise at least 14 or at least 16 or at least 18 or at
least 20 contiguous bases from SEQ ID NO: 32.
[0078] Bombyx transposons are transposable by Bombyx transposases,
for example by at least one polypeptide selected from SEQ ID NO:
407, or 412-697, optionally fused to a heterologous nuclear
localization signal. Operability of a Bombyx transposon can be
shown by the ability of a transposase having the amino acid
sequence of SEQ ID NO:415 fused to a heterologous NLS to transpose
the transposon.
[0079] Cells whose genomes contain a Bombyx transposon are an
aspect of the invention. The cell may be any eukaryotic cell.
5.2.4 Modified Transposon Target Sequences
[0080] Having observed that the natural Bombyx and Xenopus
transposons were flanked by different target sequences (5'-TTAT-3'
and 5'TTAA-3' respectively), we attempted to modify the target
sequences of piggyBac-like transposons by changing the sequence
adjacent to the ITR. This is expected to change the 5' overhangs of
the excised transposon (Mitra et al., 2008. EMBO J. 27: 1097-1109
"piggyBac can bypass DNA synthesis during cut and paste
transposition"). We created a piggyBac-TTAT transposon by joining a
5'-TTAT-3' target sequence to piggyBac left transposon end SEQ ID
NO 37 and placing this on one side of reporter construct SEQ ID NO
39, and joining piggyBac right transposon end SEQ ID NO 38 followed
by target sequence 5'-TTAT-3' to the other side. We observed that
in vivo in mammalian cells, the TTAT piggyBac transposon was
integrated by the piggyBac transposase (SEQ ID NO. 698) to give
expression of the protein encoded on the transposon at comparable
levels to the TTAA piggyBac transposon (see Section 6.1.2 and
compare Table 2 rows 24 and 26).
[0081] We made a similar switch from 5'-TTAA-3' to 5'-TTAT-3'
target sequence for the Xenopus transposon. Again we observed that
in vivo in mammalian cells, the TTAT Xenopus transposon was
integrated by a Xenopus transposase fused to a heterologous nuclear
localization signal, to give expression of the protein encoded on
the transposon at comparable levels to those from the TTAA Xenopus
transposon integrated by the same transposase (see Section 6.1.2
and compare Table 2 rows 14 and 22). Thus a Xenopus transposase is
effective at transposing transposons with different target
sequences including 5'-TTAT-3' and 5'-TTAA-3' target sequences.
[0082] Finally, we also made the reverse switch for the Bombyx
transposon, changing its target sequence to TTAA. We observed that
in vivo in mammalian cells, the TTAA Bombyx transposon was
integrated by a Bombyx transposase, to give expression of the
protein encoded on the transposon at comparable levels to those
from the TTAT Bombyx transposon integrated by the same transposase
(see Section 6.1.2 and compare Table 2 rows 3 and 11). Thus a
Bombyx transposase is effective at transposing transposons with
different target sequences including 5'-TTAT-3' and 5'-TTAA-3'
target sequences.
[0083] In all cases of piggyBac-like transposons we tested
(Trichoplusi ni, Bombyx and Xenopus), the transposases excised
their transposons precisely from the DNA in which they were
originally present, leaving a single copy of the 5'-TTAA-3' or
5'-TTAT-3' target sequence that was initially present adjacent to
each of the transposon ITRs. The precise excision of all of these
transposons by their transposases is consistent with the cut and
paste mechanism described for Trichoplusi ni piggyBac.
[0084] Bombyx transposase SEQ ID NO 407 shares 36% sequence
identity with the piggyBac transposase from Trichoplusia ni;
Xenopus transposases SEQ ID NO 48 and 49 share only 23% sequence
identity with the piggyBac transposase from Trichoplusia ni;
Xenopus transposases SEQ ID NO: 48 and 49 share only 22% sequence
identity with Bombyx transposase SEQ ID NO: 407. All 3 of these
transposases are able to efficiently transpose their transposons
when the target sequence on the transposon is switched between
5'-TTAA-3' and 5'-TTAT-3' or vice versa. These data provide
evidence the target sequence for any piggyBac-like transposon can
be switched from 5'-TTAA-3' to 5'-TTAT-3' just by changing the
target sequence flanking the transposon ITRs. A transposon with
modified target sequences can be created for active transposases
with at least 23% sequence identity to the piggyBac transposase
from Trichoplusia ni (SEQ ID NO: 698), or 22% sequence identity
with Bombyx transposase SEQ ID NO: 407, or 22% sequence identity
with Xenopus transposases SEQ ID NOS: 48 or 49, as identified using
the TBLASTN algorithm, by taking functional left and right
transposon ends and changing the target sequences adjacent to the
ITRs from 5'-TTAA-3' to 5'-TTAT-3'.
[0085] Efficient integration into 5'-TTAT-3'/5'-ATAA-3' target
sequences can be advantageous, because 5'-TTAT-3' is a reverse
complement of 5'-ATAA-3' which is part of the canonical mammalian
polyA signal 5'-aATAAa-3'. Thus the 5'-TTAT-3' insertion site
targeted by the TTAT-directed transposon occurs at almost every
polyA signal. PolyA signals are associated with transcriptionally
active regions of the chromosome. Thus transposons that insert at
5'-TTAT-3' sites, including Bombyx transposons and modified Xenopus
and piggyBac transposons, are likely to yield higher expression
levels of the genes they carry than transposons that insert at
5'-TTAA-3' sites. This effect may become more pronounced with time,
since naturally transcriptionally active regions may be more
resistant to silencing effects.
[0086] Other useable target sequences for piggyBac transposons are
5'-CTAA-3', 5'-TTAG-3', 5'-ATAA-3', 5'-TCAA-3', 5'-AGTT-3',
5'-ATTA-3', 5'-GTTA-3', 5'-TTGA-3', 5'-TTTA-3', 5'-TTAC-3',
5'-ACTA-3', 5'-AGGG-3', 5'-CTAG-3', 5'-GTAA-3', 5'-AGGT-3',
5'-ATCA-3', 5'-CTCC-3', 5'-TAAA-3', 5'-TCTC-3', 5'-TGAA-3',
5'-AAAT-3', 5'-AATC-3', 5'-ACAA-3', 5'-ACAT-3', 5'-ACTC-3',
5'-AGTG-3', 5'-ATAG-3', 5'-CAAA-3', 5'-CACA-3', 5'-CATA-3',
5'-CCAG-3', 5'-CCCA-3', 5'-CGTA-3', 5'-CTGA-3', 5'-GTCC-3',
5'-TAAG-3', 5'-TCTA-3', 5'-TGAG-3', 5'-TGTT-3', 5'-TTCA-3',
5'-TTCT-3' and 5'-TTTT-3' (Li et al., 2013. Proc. Natl. Acad. Sci
vol. 110, no. 6, E478-487). This suggests that a synthetic
piggyBac-like transposon can be created by using a repeat of one of
these sequences in place of the natural 5'-TTAA-3' or 5'-TTAT-3'
target sequence flanking the transposon ITRs. For example, a Bombyx
transposon comprises a first useable target sequence, ITR sequence
SEQ ID NO: 33, a heterologous polynucleotide, a second ITR sequence
SEQ ID NO: 33 inverted in orientation relative to the first, and a
second useable target sequence, where the first and second useable
target sequences are preferably the same. A Xenopus transposon
comprises a first useable target sequence, ITR sequence SEQ ID NO:
19, a heterologous polynucleotide, a second ITR sequence SEQ ID NO:
19 inverted in orientation relative to the first, and a second
useable target sequence, where the first and second useable target
sequences are preferably the same. Cells whose genomes contain
Xenopus or Bombyx transposons are an aspect of the invention.
5.2.5 Selection Systems for Modifying Piggybac-Like
Transposases
[0087] Two properties of transposases that are of particular
interest for genomic modifications are their ability to integrate a
polynucleotide into a target genome, and their ability to precisely
excise a polynucleotide from a target genome. Both of these can be
selected for with a suitable system.
[0088] A system for selecting for the first step of transposition,
which is excision of a transposon from a first polynucleotide,
comprises the following components: (i) A first polynucleotide
encoding a first selectable marker operably linked to sequences
that cause it to be expressed in a selection host and (ii) A first
transposon comprising transposon ends recognized by the first
transposase. The first transposon is present in, and interrupts the
coding sequence of, the first selectable marker, such that the
first selectable marker is not active. The first transposon is
placed in the first selectable marker such that precise excision of
the first transposon causes the first selectable marker to be
reconstituted. Host cells that contain the first polynucleotide,
either chromosomally or extrachromosomally, can be used to screen
for transposases that can excise the first transposon.
[0089] If the first transposon comprises a second selectable
marker, operably linked to sequences that make the second
selectable marker expressible in the selection host, transposition
of the second selectable marker into the genome of the host cell
will yield a genome comprising active first and second selectable
markers. The cell will therefore grow under selective conditions
for both markers. The second selectable marker, like the first
selectable marker, may be a gene encoding an antibiotic resistance
gene, or an auxotrophic marker, or any other selectable marker.
[0090] If the first transposon comprises a first counter-selectable
marker, operably linked to sequences that make the first
counter-selectable marker expressible in the selection host,
transposition of the first counter-selectable marker into the
genome of the host cell will yield a cell with an active first
selectable marker and active first counter-selectable marker. The
cell will therefore die under restrictive conditions for the first
counter-selectable marker.
[0091] These two selection schemes may be combined by using a
second selectable marker that is also a first counter-selectable
marker. Examples of such markers include auxotrophic marker genes
in the uracil or tryptophan synthetic pathways. These genes may be
selected for by culturing cells in the absence of the nutrient, in
this case uracil or tryptophan respectively. Biosynthetic genes may
also act as counter-selectable markers if they enable a cell to
incorporate a toxic analog in place of a genuine metabolic
precursor into their molecules. Genes in the uracil biosynthetic
pathway can convert the non-toxic compound 5-fluoroorotic acid into
toxic 5-fluorouracil, thus growing cells with 5-fluoroorotic acid
is restrictive for a functional uracil pathway. Similarly,
5-fluoroanthranilic acid is converted by the tryptophan synthesis
pathway to the toxic 5-fluorotryptophan, thus growing cells with
5-fluoroanthranilic acid is restrictive for a functional tryptophan
pathway. Host cells that contain a first polynucleotide comprising
a first selectable marker interrupted by a transposon comprising a
uracil or tryptophan gene, can be used to screen simultaneously for
hyperactive and integration-deficient transposases. For example, a
polynucleotide expressible in the host cell encoding a first
transposase or a first transposase library such as a site
saturation mutagenesis library for one or more amino acid positions
is introduced into host cells containing the first polynucleotide.
These cells are the divided into two pools. The first pool is
cultured under conditions that are selective for the first
selectable marker and restrictive for the first counter-selectable
marker. The genes encoding the transposases are then isolated from
the host cells that gained the ability to grow, and transposase
genes from this first pool of cells may be analyzed to identify
amino acid changes that enhance excision activity but not
integration activity. The second pool is cultured under conditions
that are selective for the first selectable marker and for the
second selectable marker. The genes encoding the transposases are
then isolated from the host cells that gained the ability to grow,
and transposase genes from this second pool of cells may be
analyzed to identify amino acid changes that enhance the complete
transposase activity.
[0092] These selection systems may be used to identify transposases
with modified activities by screening libraries of variant
transposases. One type of library is a pool of polynucleotides
encoding all possible amino acid substitutions at a first amino
acid position in the transposase. A site-saturation mutagenesis
library at a single position encodes twenty different polypeptides,
including one that is the natural transposase sequence. For a
transposase that is 600 amino acids long, all possible single amino
acid substitutions are present in 600 such site-saturation
mutagenesis libraries, one for each position. These libraries can
be tested using a transposase selection system to identify active
substitutions at each position.
[0093] Individual favorable mutations may be combined in a variety
of different ways, for example by "DNA shuffling" or by methods
described in U.S. Pat. No. 8,635,029 B2. A transposase with
modified activity, either for activity on a new target sequence
including a 5'-TTAT-3' target sequence, or increased activity on an
existing target sequence may be obtained by using variations of the
selection scheme outlined above with an appropriate corresponding
transposon.
[0094] Activity of transposases may also be increased by fusion of
nuclear localization signal (NLS) at the N-terminus, C-terminus,
both at the N- and C-termini or internal regions of the transposase
protein, as long as transposase activity is retained. A nuclear
localization signal or sequence (NLS) is an amino acid sequence
that `tags` or facilitates interaction of a protein, either
directly or indirectly with nuclear transport proteins for import
into the cell nucleus. Nuclear localization signals (NLS) used can
include consensus NLS sequences, viral NLS sequences, cellular NLS
sequences, and combinations thereof.
[0095] Transposases may also be fused to other protein functional
domains. Such protein functional domains can include DNA binding
domains, flexible hinge regions that can facilitate one or more
domain fusions, and combinations thereof. Fusions can be made
either to the N-terminus, C-terminus, or internal regions of the
transposase protein so long as transposase activity is retained.
DNA binding domains used can include a helix-turn-helix domain,
Zn-finger domain, a leucine zipper domain, or a helix-loop-helix
domain. Specific DNA binding domains used can include a Gal4 DNA
binding domain, a LexA DNA binding domain, or a Zif268 DNA binding
domain. Flexible hinge regions used can include glycine/serine
linkers and variants thereof.
[0096] A comparable process may be used to increase the
transposability of the transposon ends by a transposase. In this
case, the transposon may comprise a first active selectable marker.
Transposon ends may be selected from any piggyBac-like transposon.
The sequence of one or both transposon ends may be subjected to
random or pre-determined sequence changes, including changes to the
target sequence, the ITR or to other parts of the transposon ends.
The transposon may then be introduced into a first cell that
contains a target polynucleotide comprising a second active
selectable marker and an active transposase. If the transposase is
able to transpose the transposon, some fraction of the transposons
will be transposed into the target polynucleotide. The target
polynucleotide is purified from the first cell, and introduced into
a second cell which is subjected to restrictive conditions for
which it requires the first selectable marker and the second
selectable marker to survive. The transposon may be recovered, for
example by sequencing out from the transposon to identify the
flanking sequence, and then amplifying the transposon using PCR.
The process may be performed in pools of variants: a more active
transposon will create target polynucleotides containing both
selectable markers more frequently, and will thus be more highly
represented in the population. In this process, the transposon may
optionally be present as a reversible interruption in a selectable
marker as described for the transposase activity screen. However,
this is not necessary for the transposon activity screen, since the
transposed transposons are detected directly.
5.2.6. Modified Xenopus Transposases
[0097] We subjected Xenopus transposase SEQ ID NO: 48 to saturation
mutagenesis as described in Example 6.3.1.1, and identified 1,793
(16.0%) amino acid substitutions that were associated with
increased transposition activity (a composite measure of
integration and excision), and 1,074 (9.6%) amino acid
substitutions that were associated with increased excision
activity, out of a total of 11,172 possible substitutions (19
possible substitutions at each of the 588 amino acids excluding the
invariant N-terminal methionine). The two classes of substitutions
had some overlap but were neither identical nor did one class
completely contain the other. These beneficial substitutions are
shown in Table 4 columns C and D.
[0098] A similar number of substitutions were found to be
essentially neutral as to effect on transposition or excision
activity: that is, they were present at approximately the same
frequency in unselected and post-selection libraries. Thus Xenopus
transposases readily accept many amino acid substitutions without
significant functional detriment.
[0099] Xenopus transposases can thus be created that are not
naturally occurring sequences, (e.g. not SEQ ID NO: 48 or 49), but
that are at least 99% identical, or at least 98% identical, or at
least 97% identical, or at least 96% identical, or at least 95%
identical, or at least 90% identical, or at least 84% identical to
SEQ ID NO 48. Such variants can retain partial activity of the
transposase of SEQ ID NO:48 (as determined by either or both of
transposition and/or excision activity), can be functionally
equivalent of the transposase of SEQ ID NO:48 in either or both of
transposition and excision, or can have enhanced activity relative
to the transposase of SEQ ID NO:48 in transposition, excision
activity or both. Such variants can include mutations shown herein
to increase transposition and/or excision, mutations shown herein
to be neutral as to transposition and/or excision, and mutations
detrimental to transposition and/or integration or any combination
of such mutations. Preferred variants include mutations shown to be
neutral or to enhance transposition/and or excision. Some such
variants lack mutations shown to be detrimental to transposition
and/or excision. Some such variants include only mutations shown to
enhance transposition, only mutations shown to enhance excision, or
mutations shown to enhance both transposition and excision.
[0100] Enhanced activity means activity (e.g., transposition or
excision activity) that is greater beyond experimental error than
that of a reference transposase from which a variant was derived.
The activity can be greater by a factor of e.g., 1.5, 2, 5, 10, 20,
50 or 100 fold of the reference transposase. The enhanced activity
can lie within a range of for example 2-100 fold, 2-50 fold, 5-50
fold or 2-10 fold of the reference transposase. Here and elsewhere
activities can be measured as demonstrated in the examples.
[0101] Functional equivalence means a variant transposase can
mediate transposition and/or excision of the same transposon with a
comparable efficiency (within experimental error) to a reference
transposase. More than 80 representative sequences of variant
Xenopus transposases with transposition frequencies comparable to
naturally occurring Xenopus transposase SEQ ID NO 48 are SEQ ID
NOs: 325-402.
[0102] Furthermore, variant sequences of SEQ ID NO 48 can be
created by combining two, or three or four, or five or more
substitutions selected from Table 4 column C or D. Combining
beneficial substitutions, for example those shown in column C or D
of Table 4 can result in hyperactive variants of SEQ ID NO 48. Such
variants may be created in a library, for example by DNA shuffling,
and then identified by selection using a scheme as outlined in
Section 5.2.5 or Example 6.3.1. Alternatively, methods described in
U.S. Pat. No. 8,635,029 can be used to design, synthesize and test
small numbers of variants incorporating amino acid substitutions to
obtain transposases with improved integration or excision
activities.
[0103] Xenopus transposase variants that are hyperactive for
integration in yeast and mammalian cells were prepared as described
in Example 6.3.1.1. We identified at least 25 Xenopus transposases
(SEQ ID NOs: 52-76) with transposition frequencies about at least
50-fold greater than that of naturally occurring Xenopus
transposase SEQ ID NO: 48. We identified more than 130 Xenopus
transposases (SEQ ID NOs: 77-210) with transposition frequencies
between about 10-fold greater and 50-fold greater than that of
naturally occurring Xenopus transposase SEQ ID NO: 48. We
identified more than 100 Xenopus transposases (SEQ ID NOs: 211-324)
with transposition frequencies between about 2-fold greater and
10-fold greater than that of naturally occurring Xenopus
transposase SEQ ID NO: 48. These transposases comprised one or more
of the substitutions (relative to SEQ ID NO: 48) listed in Table 4
columns C and D. Preferred hyperactive Xenopus transposases
comprised one or more of the substitutions (relative to SEQ ID NO:
48) listed in Table 11 column C. Preferred hyperactive Xenopus
transposases include polypeptides comprising one of SEQ ID NOS:
52-402; some hyperactive transposases may further comprise a
heterologous nuclear localization sequence.
[0104] Preferred hyperactive Xenopus transposases comprise an amino
acid sequence, other than a naturally occurring protein (e.g., not
a transposase whose amino acid sequence comprises SEQ ID NO:48 or
49), that is at least 85% identical or at least 90% identical or at
least 95% identical, or at least 99% identical to the amino acid
sequence of any of SEQ ID NOs: 51-406, including SEQ ID NO: 61.
Some preferred hyperactive transposases comprise an amino acid
sequence, other than a naturally occurring protein, that is at
least 85% identical or at least 90% identical or at least 95%
identical, or at least 99% identical to the amino acid sequence of
SEQ ID NO: 61 and that comprises at least one amino acid
substitution (relative to SEQ ID NO: 48) shown in Table 4 column C,
Table 4 column D or Table 11 column C. Preferred hyperactive
Xenopus transposases include polypeptides comprising an amino acid
substitution at a position selected from amino acid 6, 7, 16, 19,
20, 21, 22, 23, 24, 26, 28, 31, 34, 67, 73, 76, 77, 88, 91, 141,
145, 146, 148, 150, 157, 162, 179, 182, 189, 192, 193, 196, 198,
200, 210, 212, 218, 248, 263, 270, 294, 297, 308, 310, 333, 336,
354, 357, 358, 359, 377, 423, 426, 428, 438, 447, 447, 450, 462,
469, 472, 498, 502, 517, 520, 523, 533, 534, 576, 577, 582, 583 or
587 (relative to SEQ ID NO: 48). Preferred hyperactive Xenopus
transposases include polypeptides comprising an amino acid
substitution, relative to SEQ ID NO: 48, selected from Y6C, S7G,
M16S, S19G, S20Q, S20G, S20D, E21D, E22Q, F23T, F23P, S24Y, S26V,
S28Q, V31K, A34E, L67A, G73H, A76V, D77N, P88A, N91D, Y141Q, Y141A,
N145E, N145V, P146T, P146V, P146K, P148T, P148H, Y150G, Y150S,
Y150C, H157Y, A162C, A179K, L182I, L182V, T189G, L192H, S193N,
S193K, V196I, S198G, T200W, L210H, F212N, N218E, A248N, L263M,
Q270L, S294T, T297M, S308R, L310R, L333M, Q336M, A354H, C357V,
L358F, D359N, L377I, V423H, P426K, K428R, S438A, T447G, T447A,
L450V, A462H, A462Q, I469V, I472L, Q498M, L502V, E517I, P520D,
P520G, N523S, I533E, D534A, F576R, F576E, K577I, I582R, Y583F,
L587Y or L587W, or any combination thereof including at least 1, 2,
3, 4, 5, 6, 7, 8, 9, 10 or all of these mutations.
[0105] Xenopus transposase variants that are active for excision
but deficient in integration in yeast and mammalian cells were
prepared as described in Example 6.3.1.1. Preferred
integration-deficient Xenopus transposase sequences include SEQ ID
NOS: 51 and 403-406; these integration deficient Xenopus
transposases may further comprise a heterologous nuclear
localization sequence. Preferred integration-deficient Xenopus
transposases comprise an amino acid sequence, other than a
naturally occurring protein, that is 90% identical to the amino
acid sequence of SEQ ID NO 405. Some preferred
integration-deficient transposases comprise an amino acid sequence,
other than a naturally occurring protein, that comprises an amino
acid substitution (relative to SEQ ID NO 48) wherein the Asn at
amino acid position 218 is replaced with a Glu or an Asp residue
(N218D or N218E).
[0106] Methods of creating transgenic cells using hyperactive
Xenopus transposases are an aspect of the invention. A method of
creating a transgenic cell comprises (i) introducing into a
eukaryotic cell a hyperactive Xenopus transposase (as a protein or
as a polynucleotide encoding the transposase) and a corresponding
Xenopus transposon; (ii) identifying a cell in which a Xenopus
transposon is incorporated into the genome of the eukaryotic cell.
Identifying the cell in which a Xenopus transposon is incorporated
into the genome of the eukaryotic cell may comprise selecting the
eukaryotic cell for a selectable marker encoded on the Xenopus
transposon. The selectable marker may be any selectable
polypeptide, including any described herein.
5.2.7. Modified Bombyx Transposases
[0107] We subjected Bombyx transposase SEQ ID NO 407 to saturation
mutagenesis as described in Example 6.3.1, and identified 1,176
amino acid substitutions (10.1%) that were associated with
increased transposition activity, and 1,044 (9.0%) amino acid
substitutions that were associated with increased excision
activity, out of a total of 11,571 possible substitutions (19
possible substitutions at each of the 609 amino acids excluding the
invariant N-terminal methionine). The two classes of substitutions
had some overlap but were neither identical nor did one class
completely contain the other. These beneficial substitutions are
shown in Table 4 columns G and H.
[0108] A similar number of substitutions were found to be
essentially neutral as to transposition or excision activity: that
is, they were present at approximately the same frequency in
unselected and post-selection libraries. Thus Bombyx transposases
readily accept many amino acid substitutions without significant
functional detriment. Transposases can thus be created that are not
naturally occurring sequences, e.g., not Bombyx transposase SEQ ID
NO 407, but that are at least 99% identical, or at least 98%
identical, or at least 97% identical, or at least 96% identical, or
at least 95% identical, or at least 90% identical, or at least 84%
identical to SEQ ID NO 407 (but do not comprise SEQ ID NO:407 per
se).
[0109] Such variants can retain partial activity of the transposase
of SEQ ID NO:407 (transposition and/or excision activity), can be
functionally equivalent of the transposase of SEQ ID NO:407 in
either or both of transposition and excision activity, or can have
enhanced activity relative to the transposase of SEQ ID NO:407 in
transposition, excision activity or both. Such variants can include
mutations shown herein to increase transposition and/or excision,
mutations shown herein to be neutral as to transposition and/or
excision, and mutations detrimental to transposition and/or
integration or any combination of such mutations. Preferred
variants include mutations shown to be neutral or enhancing of
transposition/and or excision. Some such variants lack mutations
shown to be detrimental to transposition and/or excision. Some such
variants include only mutations shown to enhance transposition,
only mutations shown to enhance excision, or mutations shown to
enhance both transposition and excision
[0110] Enhanced activity means activity that is greater beyond
experimental error of that of a reference transposase from which a
variant was derived. The activity can be greater by a factor of
e.g., 1.5, 2, 5, 10, 20, 50 or 100 fold of the reference
transposase. The enhanced activity can lie within a range of for
example 2-100 fold, 2-50 fold, 5-50 fold or 2-10 fold of the
reference transposase. Here and elsewhere activities can be
measured as demonstrated in the examples.
[0111] More than 60 representative sequences of variant Bombyx
transposases with transposition frequencies comparable to naturally
occurring Bombyx transposase SEQ ID NO 407 are SEQ ID NOs:
634-697.
[0112] Furthermore, variant sequences of SEQ ID NO: 407 can be
created by combining two, or three or four, or five or more
substitutions shown in Table 4 columns G and H. Combining
beneficial substitutions, for example those shown in column G or H
of Table 4 can result in hyperactive variants of SEQ ID NO: 407.
Such variants may be created in a library, for example by DNA
shuffling, and then identified by selection using a scheme as
outlined in Section 5.2.5 or Example 6.3.1.
[0113] Bombyx transposase variants that are hyperactive for
integration in yeast and mammalian cells were prepared as described
in Example 6.3.2.1. Many hyperactive transposases were obtained. We
identified at least 20 Bombyx transposases (SEQ ID NOs: 412-431)
with transposition frequencies about at least 50-fold greater than
that of naturally occurring Bombyx transposase SEQ ID NO: 407. We
identified more than 90 Bombyx transposases (SEQ ID NOs: 432-524)
with transposition frequencies between about 10-fold greater and
50-fold greater than that of naturally occurring Bombyx transposase
SEQ ID NO: 407. We identified more than 100 Bombyx transposases
(SEQ ID NOs: 525-633) with transposition frequencies between about
2-fold greater and 10-fold greater than that of naturally occurring
Bombyx transposase SEQ ID NO: 407. These transposases comprised one
or more of the substitutions (relative to SEQ ID NO: 407) listed in
Table 4 columns G and H. Preferred hyperactive Bombyx transposases
comprise one or more of the substitutions (relative to SEQ ID NO:
407) listed in Table 4 columns G and H or Table 11 column H.
Preferred hyperactive Bombyx transposases include polypeptides
comprising one of SEQ ID NOS: 412-524; these hyperactive
transposases may further comprise a heterologous nuclear
localization sequence. Preferred hyperactive transposases comprise
an amino acid sequence, other than a naturally occurring protein,
that is at least 85% identical or at least 90% identical or at
least 95% identical, or at least 99% identical to the amino acid
sequence of SEQ ID NO: 415. Preferred hyperactive Bombyx
transposases include polypeptides comprising an amino acid
substitution at a position selected from 92, 93, 96, 97, 165, 178,
189, 196, 200, 201, 211, 215, 235, 238, 246, 253, 258, 261, 263,
271, 303, 321, 324, 330, 373, 389, 399, 402, 403, 404, 448, 473,
484, 507, 523, 527, 528, 543, 549, 550, 557, 601, 605, 607, 609 or
610 (relative to SEQ ID NO: 407). Preferred hyperactive Bombyx
transposases include polypeptides comprising an amino acid
substitution, relative to SEQ ID NO: 407, selected from Q92A, V93L,
V93M, P96G, F97H, F97C, H165E, H165W, E178S, E178H, C189P, A196G,
L200I, A201Q, L211A, W215Y, G219S, Q235Y, Q235G, Q238L, K246I,
K253V, M258V, F261L, S263K, C271S, N303R, F321W, F321D, V324K,
V324H, A330V, L373C, L373V, V389L, S399N, R402K, T403L, D404Q,
D404S, D404M, N441R, G448W, E449A, V469T, C473Q, R484K, T507C,
G523A, I527M, Y528K, Y543I, E549A, K550M, P557S, E601V, E605H,
E605W, D607H, S609H or L610I, and any combination thereof. Some
combinations include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or all
of these mutations. Some preferred hyperactive transposases
comprise an amino acid sequence, other than a naturally occurring
protein, that is at least 85% identical or at least 90% identical
or at least 95% identical, or at least 99% identical to the amino
acid sequence of SEQ ID NO: 415 and that comprises at least one
amino acid substitution (relative to SEQ ID NO: 407) shown in Table
4 column F, Table 4, G or Table 11 column H.
[0114] Bombyx transposase variants that are active for excision but
deficient in integration in yeast and mammalian cells were prepared
as described in Example 6.3.2.1. Preferred integration-deficient
transposase sequences comprise one of SEQ ID NOS: 409-411; these
integration deficient transposases may further comprise a
heterologous nuclear localization sequence. Preferred
integration-deficient transposases comprise an amino acid sequence,
other than a naturally occurring protein, that is at least 90%
identical to the amino acid sequence of SEQ ID NO: 411.
[0115] Methods of creating transgenic cells using hyperactive
Bombyx transposases are an aspect of the invention. A method of
creating a transgenic cell comprises (i) introducing into a
eukaryotic cell a hyperactive Bombyx transposase (as a protein or
as a polynucleotide encoding the transposase) and a corresponding
Bombyx transposon; (ii) identifying a cell in which a Bombyx
transposon is incorporated into the genome of the eukaryotic cell.
Identifying the cell in which a Bombyx transposon is incorporated
into the genome of the eukaryotic cell may comprise selecting the
eukaryotic cell for a selectable marker encoded on the Bombyx
transposon. The selectable marker may be any selectable
polypeptide, including any described herein.
5.2.8 Gene Transfer Systems
[0116] Gene transfer systems comprise a polynucleotide to be
transferred to a host cell. The gene transfer system may comprise
any of the transposons or transposases described herein, or it may
comprise one or more polynucleotides that have other features that
facilitate efficient gene transfer without the need for a
transposase or transposon.
[0117] When there are multiple components of a gene transfer
system, for example the one or more polynucleotides comprising
genes for expression in the target cell and optionally comprising
transposon ends, and a transposase (which may be provided either as
a protein or encoded by a nucleic acid), these components can be
transfected into a cell at the same time, or sequentially. For
example, a transposase protein or its encoding nucleic acid may be
transfected into a cell prior to, simultaneously with or subsequent
to transfection of a corresponding transposon. Additionally,
administration of either component of the gene transfer system may
occur repeatedly, for example, by administering at least two doses
of this component.
[0118] Bombyx or Xenopus transposase proteins may be encoded by
polynucleotides including RNA or DNA. Preferable RNA molecules
include those with appropriate substitutions to reduce toxicity
effects on the cell, for example substitution of uridine with
pseudouridine, and substitution of cytosine with 5-methyl cytosine.
Similarly, the nucleic acid encoding the transposase protein or the
transposon of this invention can be transfected into the cell as a
linear fragment or as a circularized fragment, either as a plasmid
or as recombinant viral DNA.
[0119] The components of the gene transfer system may be
transfected into one or more cells by techniques such as particle
bombardment, electroporation, microinjection, combining the
components with lipid-containing vesicles, such as cationic lipid
vesicles, DNA condensing reagents (example, calcium phosphate,
polylysine or polyethyleneimine), and inserting the components
(that is the nucleic acids thereof into a viral vector and
contacting the viral vector with the cell. Where a viral vector is
used, the viral vector can include any of a variety of viral
vectors known in the art including viral vectors selected from the
group consisting of a retroviral vector, an adenovirus vector or an
adeno-associated viral vector. The gene transfer system may be
formulated in a suitable manner as known in the art, or as a
pharmaceutical composition or kit.
5.2.9 Sequence Elements in Gene Transfer Systems
[0120] Expression of genes from a gene transfer polynucleotide
integrated into a host cell genome is often strongly influenced by
the chromatin environment into which it integrates. Polynucleotides
that are integrated into euchromatin have higher levels of
expression than those that are either integrated into
heterochromatin, or which become silenced following their
integration. Silencing of a heterologous polynucleotide may be
reduced if it comprises a chromatin control element. It is thus
advantageous for gene transfer systems to comprise chromatin
control elements such as sequences that prevent the spread of
heterochromatin (insulators). For example, it is advantageous for a
gene transfer polynucleotide that will be integrated into a host
genome to comprise a sequence that is at least 95% identical to a
sequence selected from one of SEQ ID NOS: 869-876 and SEQ ID NO:
866. Advantageous gene transfer polynucleotides comprise an
insulator sequence that is at least 95% identical to a sequence
selected from one of SEQ ID NOS: 859-865, they may also comprise
ubiquitously acting chromatin opening elements (UCOEs) or
stabilizing and anti-repressor elements (STARs), to increase
long-term stable expression from the integrated gene transfer
polynucleotide.
[0121] In some cases, it is advantageous for a gene transfer
polynucleotide to comprise two insulators, one on each side of the
heterologous polynucleotide that contains the sequences to be
expressed. The insulators may be the same, or they may be
different. Particularly advantageous gene transfer polynucleotides
comprise a sequence that is at least 95% identical to a sequence
selected from one of SEQ ID NO: 864 or SEQ ID NO: 865 and a
sequence that is at least 95% identical to a sequence selected from
one of SEQ ID NOS: 859-865. Insulators also shield expression
control elements from one another. For example, when a gene
transfer polynucleotide comprises genes encoding two open reading
frames, each operably linked to a different promoter, one promoter
may reduce expression from the other in a phenomenon known as
transcriptional interference. Interposing an insulator sequence
that is at least 95% identical to a sequence selected from one of
SEQ ID NOS: 859-865 between the two transcriptional units can
reduce this interference, and increase expression from one or both
promoters.
[0122] In preferred embodiments, a gene transfer vector comprises
expression elements capable of driving high levels of gene
expression. In eukaryotic cells, gene expression is regulated by
several different classes of elements, including enhancers,
promoters, introns, RNA export elements, polyadenylation sequences
and transcriptional terminators.
[0123] Particularly advantageous gene transfer polynucleotides for
the transfer of genes for expression into mammalian cells comprise
an enhancer for immediate early genes 1, 2 and 3 of cytomegalovirus
(CMV) from either human or murine cells (for example sequences at
least 95% identical to any of SEQ ID NOS: 877-889), an enhancer
from the adenoviral major late protein enhancer (for example
sequences at least 95% identical to SEQ ID NO: 890), or an enhancer
from SV40 (for example sequences at least 95% identical to SEQ ID
NO: 891).
[0124] Particularly advantageous gene transfer polynucleotides for
the transfer of genes for expression into mammalian cells comprise
an EF1a promoter from any mammalian or avian species including
human, rat, mice, chicken and Chinese hamster, (for example
sequences at least 95% identical to any of SEQ ID NOS: 892-910); a
promoter from the immediate early genes 1, 2 and 3 of
cytomegalovirus (CMV) from either human or murine cells (for
example sequences at least 95% identical to any of SEQ ID NOS:
911-920); a promoter for eukaryotic elongation factor 2 (EEF2) from
any mammalian or avian species including human, rat, mice, chicken
and Chinese hamster, (for example sequences at least 95% identical
to any of SEQ ID NOS: 921-928); a GAPDH promoter from any mammalian
or yeast species (for example sequences at least 95% identical to
any of SEQ ID NOS: 936 and 949-951), an actin promoter from any
mammalian or avian species including human, rat, mice, chicken and
Chinese hamster (for example sequences at least 95% identical to
any of SEQ ID NOS: 929-935); a PGK promoter from any mammalian or
avian species including human, rat, mice, chicken and Chinese
hamster (for example sequences at least 95% identical to any of SEQ
ID NOS: 937-940), or a ubiquitin promoter (for example sequences at
least 95% identical to SEQ ID NO: 941).
[0125] Particularly advantageous gene transfer polynucleotides for
the transfer of genes for expression into mammalian cells comprise
an intron from immediate early genes 1, 2 and 3 of cytomegalovirus
(CMV) from either human or murine cells (for example sequences at
least 95% identical to any of SEQ ID NOS: 958-965), an intron from
EF1a from any mammalian or avian species including human, rat,
mice, chicken and Chinese hamster, (for example sequences at least
95% identical to any of SEQ ID NOS: 970-976), an intron from EEF2
from any mammalian or avian species including human, rat, mice,
chicken and Chinese hamster, (for example sequences at least 95%
identical to any of SEQ ID NOS: 989-996); an intron from actin from
any mammalian or avian species including human, rat, mice, chicken
and Chinese hamster (for example sequences at least 95% identical
to any of SEQ ID NOS: 977-985), a GAPDH intron from any mammalian
or avian species including human, rat, mice, chicken and Chinese
hamster (for example sequences at least 95% identical to any of SEQ
ID NOS: 986 or 987); an intron comprising the adenoviral major late
protein enhancer for example sequences at least 95% identical to
SEQ ID NO: 988) or a hybrid/synthetic intron (for example sequences
at least 95% identical to any of SEQ ID NOS: 966-969).
[0126] Particularly advantageous gene transfer polynucleotides
comprise combinations of promoters and introns in which a promoter
from one gene is combined with an intron for a different gene, that
is the intron is heterologous to the promoter. For example, an
immediate early CMV promoter from mouse (e.g. SEQ ID NOS: 916-920)
or from human (for example, SEQ ID NOS: 912-915) is advantageously
followed by an intron from EF1a (e.g. SEQ ID NOS: 970-976) or an
intron from EEF2 (for example, SEQ ID NOS: 989-996).
[0127] Particularly advantageous gene transfer polynucleotides for
the transfer of genes for expression into mammalian cells comprise
one or more of an expression enhancer that enhances RNA export from
the nucleus such as woodchuck hepatitis post-transcriptional
regulatory element (WPRE) or hepatitis B virus post-transcriptional
regulatory element (HPRE) (for example sequences at least 95%
identical to any of SEQ ID NOS: 867 or 868) and elements such as
scaffold attachment region (SAR) sequences (for example sequences
at least 95% identical to any of SEQ ID NOS: 869-876). These
expression enhancing elements are particularly advantageous when
placed 3' of a sequence to be expressed. We have determined that
SAR sequences SEQ ID NOs: 869-871 enhance expression of an open
reading frame more when they are within the transcript than when
they are after the polyadenylation signal. This is unexpected,
since the proposed role of SARs is in attaching the genomic DNA
sequences to the nuclear scaffold. SAR SEQ ID NOs: 869-871 are
particularly beneficial to expression of a polypeptide when
combined with naturally occurring HPRE post-transcriptional
regulatory element SEQ ID NO: 868, for example as in SEQ ID NO:
866. They are equally beneficial when combined with a modified
variant of HPRE post-transcriptional regulatory element SEQ ID NO:
867, which we made by introducing a matched pair of mutations to
remove a BfuAI restriction site without altering the RNA stem-loop
structure of the element, for example as in SEQ ID NO: 1100. We
tested the expression-enhancing effects of SEQ ID NO: 1100, by
comparing expression of a gene encoding DasherGFP from
polynucleotides that comprised either SEQ ID NO: 866 or SEQ ID NO:
1100 or no additional expression enhancing elements between the
DasherGFP gene and the rabbit globin polyA sequence. The
polynucleotides were integratred into the genome of CHO cells, and
expression of DasherGFP measured. SEQ ID NO: 866 and SEQ ID NO:
1100 both produced expression levels of Dasher GFP that were at
least 110% or at least 120% or at least 200% or at least 500% of
the expression achieved without either element. Advantageous gene
transfer polynucleotides comprise a sequence that is at least 95%
identical or at least 98% identical or at least 99% identical or at
least 99.5% identical to SEQ ID NO: 866, or a sequence that is
either SEQ ID NO: 866 or SEQ ID NO: 1100. These are particularly
beneficial when further combined with a strong polyadenylation
signal sequence, for example the signal from the rabbit beta globin
gene, for example as in SEQ ID NO: 1101-2. The effects of these
elements may be further enhanced when combined with an insulator
sequence. Particularly advantageous combinations are given as SEQ
ID NO: 820-858. An advantageous gene transfer polynucleotide
comprises a sequence that is at least 90% identical or at least 95%
identical or at least 99% identical with any of SEQ ID NO: 820-858.
Particularly advantageous gene transfer polynucleotides comprise a
Xenopus or Bombyx transposon comprising a sequence that is at least
90% identical or at least 95% identical or at least 99% identical
to a sequence selected from SEQ ID NO: 820-858.
[0128] Particularly advantageous gene transfer polynucleotides for
the transfer of a first and a second gene for co-expression into
mammalian cells comprise a promoter and optionally enhancer and
introns operably linked to the first gene, and a translational
coupling element such as an IRES operably linking expression of a
second gene to the first. Particularly advantageous gene transfer
polynucleotides comprise an IRES sequence selected from SEQ ID NOS:
1050-1094.
[0129] Expression of two genes from a single polynucleotide can
also be accomplished by operably linking the expression of each
gene to a separate promoter, each of which may optionally be
operably linked to enhancers and introns as described above. It is
often advantageous to place a genetic insulator such as the HS4
core or D4Z4 core, between the two promoters, for example after the
polyadenylation sequence operably linked to the gene encoding the
first polypeptide and before the promoter operably linked to the
gene encoding the second polypeptide. See Example 6.2.1 and Table
7, and compare row 12 with row 13, row 14 with row 15, row 16 with
row 17, row 18 with row 19, row 20 with row 21 and row 22 with row
23. In each case the expression of the first polypeptide, the
second polypeptide or both polypeptides was increased by the
presence of an insulator sequence interposed between the two
promoters.
[0130] Particularly advantageous combinations of promoters for
expression of two polypeptides include configurations in which one
polypeptide is expressed operably linked to the EF1a promoter or
the CMV promoter and the second polypeptide is expressed operably
linked to the CMV promoter, the GAPDH promoter, the EF1a promoter
or the actin promoter. Specific combinations of polyadenylation
signals, terminators, enhancers, promoters, introns, 5'UTRs and
insulators sequences that work well when placed following a gene
that encodes a first polypeptide and preceding a gene that encodes
a second polypeptide (i.e. in a spacer polynucleotide) include SEQ
ID NOS: 998-1049. Particularly advantageous gene transfer
polynucleotides for the transfer of a first and a second gene for
co-expression into mammalian cells comprise a sequence at least 90%
identical or at least 95% identical or at least 99% identical to a
sequence selected from SEQ ID NOS: 998-1049.
5.2.10 Increasing Expression by Selection
[0131] High levels of expression may be obtained from genes encoded
on gene transfer polynucleotides that are integrated at regions of
the genome that are highly transcriptionally active, or that are
integrated into the genome in multiple copies, or that are present
extrachromosomally in multiple copies.
[0132] The expression of a first expression polypeptide encoded on
a gene transfer polynucleotide (the "expression polypeptide") can
be increased if the gene transfer polynucleotide also comprises a
sequence encoding a selectable polypeptide. It is often
advantageous to operably link the gene encoding the selectable
polypeptide to expression control elements that result in low
levels of expression of the selectable polypeptide from the gene
transfer polynucleotide and/or to use conditions that provide more
stringent selection. Under these conditions, for the expression
cell to produce sufficient levels of the selectable polypeptide
encoded on the gene transfer polynucleotide to survive the
selection conditions, the gene transfer polynucleotide must either
be present in a favorable location in the cell's genome for high
levels of expression, or a sufficiently high number of copies of
the gene transfer polynucleotide must be present, such that these
factors compensate for the low levels of expression achievable
because of the expression control elements.
[0133] The expression polypeptide and the selectable polypeptide
may be included on the same gene transfer polynucleotide, but
operably linked to different promoters. In this case low expression
levels of the selectable marker may be achieved by using a weakly
active constitutive promoter such as the phosphoglycerokinase (PGK)
promoter (e.g. SEQ ID NOS: 937-940), the Herpes Simplex Virus
thymidine kinase (HSV-TK) promoter (e.g. SEQ ID NO: 943), the MCI
promoter (for example SEQ ID NO: 944), the ubiquitin promoter (for
example SEQ ID NO: 941). Other weakly active promoters maybe
deliberately constructed, for example a promoter attenuated by
truncation, such as a truncated SV40 promoter (for example SEQ ID
NO: 945 or 946), a truncated HSV-TK promoter (for example SEQ ID
NO: 942), or a promoter attenuated by insertion of a 5'UTR
unfavorable for expression between a promoter and the gene encoding
the selectable polypeptide, for example SEQ ID NOS: 956 or 957.
Examples of attenuated promoters include an attenuated PGK promoter
(SEQ ID NO: 947) and an attenuated HSV-TK promoter (SEQ ID NO:
948). Particularly advantageous gene transfer polynucleotides
comprise a sequence that is at least 90% identical or at least 95%
identical or at least 99% identical to any of SEQ ID NOS: 937-948,
operably linked to a gene encoding a selectable marker.
[0134] Expression levels of a selectable marker may also be
advantageously reduced by other mechanisms such as the insertion of
the SV40 small t antigen intron after the gene for the selectable
marker. The SV40 small t intron accepts aberrant 5' splice sites,
and can lead to deletions within the preceding gene in a fraction
of the spliced mRNAs, thereby reducing expression of the selectable
marker. Particularly advantageous gene transfer polynucleotides
comprise intron SEQ ID NO:997, operably linked to a gene encoding a
selectable marker. For this mechanism of attenuation to be
effective, it is preferable for the gene encoding the selectable
marker to comprise a strong intron donor within its coding region.
Glutamine synthase SEQ ID NO: 703 may be encoded by the sequence
SEQ ID NO: 704 which comprises a strong intron donor. Puromycin
acetyl transferase SEQ ID NO: 715 may be encoded by the sequence
SEQ ID NO: 716 which comprises a strong intron donor. Particularly
advantageous gene transfer polynucleotides comprise a sequence at
least 90% identical or at least 95% identical or at least 99%
identical to either of SEQ ID NO: 704 or SEQ ID NO: 716, and SEQ ID
NO:997.
[0135] Expression levels of a selectable marker may also be
advantageously reduced by other mechanisms such as insertion of an
inhibitory 5'-UTR within the transcript, for example SEQ ID NO: 956
or 957. Particularly advantageous gene transfer polynucleotides
comprise a promoter operably linked to a gene encoding a selectable
marker, wherein a sequence that is at least 90% identical or at
least 95% identical or at least 99% identical to SEQ ID NO: 956 or
957 is interposed between the promoter and the selectable
marker.
[0136] Table 13 shows the transposition of transposons comprising a
puromycin selectable marker operably linked to a relatively strong
PGK promoter (SEQ ID NO: 937; Table 13 rows 2-4), or to a weaker
HSV-TK promoter (SEQ ID NO: 942; Table 13 rows 5-9). Expression
from transposons in which puromycin acetyl transferase was operably
linked to the weaker promoter was substantially higher than from
transposons in which puromycin acetyl transferase was operably
linked to the stronger promoter. However, this high expression
required co-transfection of the transposon with a transposase. By
operably linking the selectable marker to elements that result in
weak expression, cells are selected which either incorporate
multiple copies of the transposon, or in which the transposon is
integrated at a favorable genomic location for high expression.
Using a gene transfer system that comprises a transposon and a
corresponding transposase, particularly a Xenopus transposon and a
hyperactive Xenopus transposase or a Bombyx transposon and a
hyperactive Bombyx transposase increases the likelihood that cells
will be produced with multiple copies of the transposon, or in
which the transposon is integrated at a favorable genomic location
for high expression, as shown in Examples 6.3.1.2 and 6.3.2.2. Gene
transfer systems comprising a transposon and a corresponding
transposase are thus particularly advantageous when the transposon
comprises a selectable marker operably linked to weak promoters,
and when the transposase is a hyperactive transposase. Particularly
advantageous transposons comprise selectable markers operably
linked to a promoter with at least 90% identity or at least 95%
identity or at least 99% identity to a sequence selected from SEQ
ID NOS: 942-948. Particularly advantageous gene transfer
polynucleotides comprise sequences with at least 90% identity or at
least 95% identity or at least 99% identity to a sequence selected
from SEQ ID NOS: 719-749.
[0137] Another way to select for high levels of expression of a
first expression polypeptide, is to translationally couple the gene
encoding a selectable marker and the first expression polypeptide
using an IRES. Preferably the IRES results in a much higher
expression of the first expression polypeptide than the selectable
marker. Many new IRES activities are shown in Table 9 and described
in Example 6.4.1. In these examples, the first expression
polypeptide is a green fluorescent protein and the selectable
polypeptide is a red fluorescent protein. Each table also shows the
expression level of the first expression polypeptide in a construct
lacking the IRES and gene for the selectable polypeptide.
Particularly desirable IRES elements are those that have a high
ratio of expression between the first expression polypeptide and
the selectable polypeptide, and that also have levels of expression
of the first expression polypeptide that are close to the levels of
expression obtained in the absence of the IRES and gene for the
selectable polypeptide. From Table 9, it can be seen that IRES SEQ
ID NOs: 1089, 1078, 1080, 1086, 1076, 1075, 1081, 1077, 1088, 1079,
1091, 1066, 1094, 1093, 1072, 1068, 1071 have levels of expression
of the first expression polypeptide that are at least 50% of the
levels of expression obtained in the absence of the IRES and a
second open reading frame in CHO cells, and IRES SEQ ID NOs: 1084,
1079, 1073, 1085, 1082, 1074, 1080, 1066 have levels of expression
of the first expression polypeptide that are at least 50% of the
levels of expression obtained in the absence of the IRES and a
second open reading frame in HEK cells. IRES SEQ ID NOs:1091, 1070,
1069, 1090, 1094, 1077, 1067, 1089, 1068, 1078, 1066, 1072, 1093,
1092, 1079, 1080, 1081, 1052, 1074, 1085, 1076, 1088, 1075 and 1086
all express the second ORF at 15% or less than the level of the
first ORF in CHO cells. IRES SEQ ID NOs: 1091, 1067, 1094, 1070,
1089, 1092, 1090, 1069, 1078, 1074, 1077, 1085, 1084, 1053, 1096
and 1073 all express the second ORF at 20% or less than the level
of the first ORF in HEK cells.
[0138] These IRES elements are therefore particularly advantageous,
when used to link the expression of a first expression polypeptide
to the expression of a gene encoding a selectable marker in a gene
transfer polynucleotide, wherein the gene transfer polynucleotide
comprises a gene encoding a first expression polypeptide on the 5'
side of the IRES and a gene encoding a selectable marker on the 3'
side of the IRES. Particularly advantageous gene transfer
polynucleotides comprise selectable markers operably linked to an
IRES selected from SEQ ID NOS: 1052, 1053, 1066, 1068, 1072, 1073,
1074, 1075, 1076, 1077, 1078, 1079, 1080, 1081, 1084, 1085, 1086,
1088, 1089, 1090, 1091, 1093 or 1094.
[0139] Common selectable polypeptides confer resistance of
eukaryotic cells to antibiotics such as neomycin (resistance
conferred by an aminoglycoside 3'-phosphotransferase e.g. SEQ ID
NO: 709-712), puromycin (resistance conferred by puromycin
acetyltransferase e.g. SEQ ID NOS: 713-716), blasticidin
(resistance conferred by a blasticidin acetyltransferase and a
blasticidin deaminase), hygromycin B (resistance conferred by
hygromycin B phosphotransferase e.g. SEQ ID NO: 717-718 and zeocin
(resistance conferred by binding protein, for example SEQ ID NO:
702). Other selectable polypeptides include those that are
fluorescent (such as GFP, RFP etc.) and can therefore be selected
for example using flow cytometry. Other selectable polypeptides
include transmembrane proteins that are able to bind to a second
molecule (protein or small molecule) that can be fluorescently
labelled so that the presence of the transmembrane protein can be
selected for example using flow cytometry.
[0140] Glutamine synthase (GS, for example SEQ ID NOS: 703 and 705)
is used as a selectable marker that allows selection via glutamine
metabolism. Glutamine synthase is the enzyme responsible for the
biosynthesis of glutamine from glutamate and ammonia, and is a
crucial component of the only pathway for glutamine formation in a
mammalian cell. In the absence of glutamine in the growth medium,
the GS enzyme is essential for the survival of mammalian cells in
culture. Some cell lines, for example mouse myeloma cells do not
express sufficient GS enzyme to survive without added glutamine. In
these cells a transfected GS gene can function as a selectable
marker by permitting growth in a glutamine-free medium. In other
cell lines, for example Chinese hamster ovary (CHO) cells express
sufficient GS enzyme to survive without exogenously added
glutamine. These cell lines can be manipulated by genome editing
techniques including CRISPR/Cas9 to reduce or eliminate the
activity of the GS enzyme. In all of these cases, GS inhibitors
such as methionine sulphoximine (MSX) can be used to inhibit a
cell's endogenous GS activity. Selection protocols include
introducing a construct comprising sequences encoding a first
polypeptide and a glutamine synthase selectable marker, and then
treating the cell with inhibitors of glutamine synthase such as
methionine sulphoximine. The higher the levels of methionine
sulphoximine that are used, the higher the level of glutamine
synthase expression is required to allow the cell to synthesize
sufficient glutamine to survive. Some of these cells will also show
an increased expression of the first polypeptide.
[0141] Preferably the GS gene is operably linked to a weak promoter
or other sequence elements that attenuate expression as described
herein, such that high levels of expression can only occur if many
copies of the gene transfer polynucleotide are present, or if they
are integrated in a position in the genome where high levels of
expression occur.
[0142] A second system for increasing expression by selection uses
the enzyme dihydrofolate reductase (DHFR, for example SEQ ID NO:
707 or 708) which is required for catalyzing the reduction of
5,6-dihydrofolate (DHF) to 5,6,7,8-tetrahydrofolate (THF) and is
used as a selectable marker. Some cell lines do not express
sufficient DHFR to survive without added THF. In these cells a
transfected DHFR gene can function as a selectable marker by
permitting growth in a THF-free medium. DHFR-deficient cell lines,
for example Chinese hamster ovary (CHO) cells can be produced by
genome editing techniques including CRISPR/Cas9 to reduce or
eliminate the activity of the endogenous DHRF enzyme. DHFR confers
resistance to methotrexate (MTX). DHFR can be inhibited by higher
levels of methotrexate. Selection protocols include introducing a
construct comprising sequences encoding a first polypeptide and a
DHFR selectable marker into a cell with or without an endogenous
DHFR gene, and then treating the cell with inhibitors of DHFR such
as methotrexate. The higher the levels of methotrexate that are
used, the higher the level of DHFR expression is required to allow
the cell to synthesize sufficient DHFR to survive. Some of these
cells will also show an increased expression of the first
polypeptide. Preferably the DHFR gene is operably linked to a weak
promoter or other sequence elements that attenuate expression as
described above, such that high levels of expression can only occur
if many copies of the gene transfer polynucleotide are present, or
if they are integrated in a position in the genome where high
levels of expression occur.
[0143] The combination of selectable marker and its operably linked
control elements profoundly affect the expression that can be
obtained from a gene transfer polynucleotide. Particularly
advantageous gene transfer polynucleotides comprise a sequence that
is at least 90% identical or at least 95% identical or at least 99%
identical to a sequence selected from SEQ ID NOS: 719-749.
Preferably these sequences are flanked by a pair of transposon
ends.
[0144] As shown in Table 15 and described in Example 6.3.2.2, the
combination of selectable marker and its operably linked control
elements profoundly affect the expression that can be obtained from
a second promoter on a gene transfer polynucleotide. These effects
are also influenced by the presence of insulator sequences on the
gene transfer polynucleotide. Particularly advantageous gene
transfer polynucleotides comprise a sequence that is at least 90%
identical or at least 95% identical or at least 99% identical to a
sequence selected from SEQ ID NOS: 751-819. Particularly
advantageous gene transfer polynucleotides comprise a Xenopus or
Bombyx transposon comprising a sequence that is at least 90%
identical or at least 95% identical or at least 99% identical to a
sequence selected from SEQ ID NOS: 751-819.
[0145] The use of transposons and transposases in conjunction with
such selectable markers that are required for normal cell
metabolism has several advantages over non-transposon constructs.
One is that linkage between expression of the first polypeptide and
the selectable marker is better for transposons, because a
transposase will integrate the entire sequence that lies between
the two transposon ends into the genome. In contrast when
heterologous DNA is introduced into the nucleus of a eukaryotic
cell, for example a mammalian cell, it is gradually broken into
random fragments which may either be integrated into the cell's
genome, or degraded. Thus if a construct comprising sequences that
encode a first polypeptide and a selectable marker required for
normal cell metabolism is introduced into a population of cells,
some cells will integrate the sequences encoding the selectable
marker but not those encoding the first polypeptide, and vice
versa. Selection of cells expressing high levels of selectable
marker is thus only somewhat correlated with cells that also
express high levels of the first polypeptide. In contrast, because
the transposase integrates all of the sequences between the
transposon ends, cells expressing high levels of selectable marker
are highly likely to also express high levels of the first
polypeptide.
[0146] A second advantage of transposons and transposases is that
they are much more efficient at integrating DNA sequences into the
genome. Thus a much higher fraction of the cell population is
likely to receive one or more copies of the construct in their
genomes, so there will be a correspondingly higher likelihood of
good stable expression of both the selectable marker and the first
polypeptide.
[0147] A transposon that comprises a sequence encoding a first
polypeptide and a selectable marker that can be inhibited by a
small molecule inhibitor may thus be used to obtain cells
expressing high levels of the first polypeptide. The first
polypeptide may be part of an antibody. Preferred selectable
markers are glutamine synthase and DHFR.
[0148] Higher numbers of integrated transposons may be selected
using selectable markers required for normal cell metabolism such
as DHFR or glutamine synthase.
5.3 Kits
[0149] The present invention also features kits comprising a Bombyx
transposase as a protein or encoded by a nucleic acid, and/or a
Bombyx transposon; or a gene transfer system as described herein
comprising a Bombyx transposase as a protein or encoded by a
nucleic acid as described herein, in combination with a Bombyx
transposon; optionally together with a pharmaceutically acceptable
carrier, adjuvant or vehicle, and optionally with instructions for
use. Any of the components of the inventive kit may be administered
and/or transfected into cells in a subsequent order or in parallel,
e.g. a Bombyx transposase protein or its encoding nucleic acid may
be administered and/or transfected into a cell as defined above
prior to, simultaneously with or subsequent to administration
and/or transfection of a Bombyx transposon. Alternatively, a Bombyx
transposon may be transfected into a cell as defined above prior
to, simultaneously with or subsequent to transfection of a Bombyx
transposase protein or its encoding nucleic acid. If transfected in
parallel, preferably both components are provided in a separated
formulation and/or mixed with each other directly prior to
administration to avoid transposition prior to transfection.
Additionally, administration and/or transfection of at least one
component of the kit may occur in a time staggered mode, e.g. by
administering multiple doses of this component.
[0150] In addition, the present invention also features kits
comprising a Xenopus transposase as a protein or encoded by a
nucleic acid, and/or a Xenopus transposon; or a gene transfer
system as described herein comprising a Xenopus transposase as a
protein or encoded by a nucleic acid as described herein, in
combination with a Xenopus transposon; optionally together with a
pharmaceutically acceptable carrier, adjuvant or vehicle, and
optionally with instructions for use. Any of the components of the
inventive kit may be administered and/or transfected into cells in
a subsequent order or in parallel, e.g. a Xenopus transposase
protein or its encoding nucleic acid may be administered and/or
transfected into a cell as defined above prior to, simultaneously
with or subsequent to administration and/or transfection of a
Xenopus transposon. Alternatively, a Xenopus transposon may be
transfected into a cell as defined above prior to, simultaneously
with or subsequent to transfection of a Xenopus transposase protein
or its encoding nucleic acid. If transfected in parallel,
preferably both components are provided in a separated formulation
and/or mixed with each other directly prior to administration the
to avoid transposition prior to transfection. Additionally,
administration and/or transfection of at least one component of the
kit may occur in a time staggered mode, e.g. by administering
multiple doses of this component.
6. EXAMPLES
[0151] The following examples illustrate the methods, compositions
and kits disclosed herein and should not be construed as limiting
in any way. Various equivalents will be apparent from the following
examples; such equivalents are also contemplated to be part of the
invention disclosed herein.
6.1 Transposases
6.1.1 Xenopus- and Bombyx-Derived Transposases
[0152] Joining a pair of transposon ends onto the ends of a
heterologous polynucleotide can create a synthetic transposon that
can be integrated into a target genome by a transposase. Table 1
shows the configurations of 4 different synthetic transposons
created by joining the transposon end whose SEQ ID NO is given in
column A to one side of the reporter construct SEQ ID NO: 39,
joining the transposon end whose SEQ ID NO is given in column B to
the other side of the reporter construct and flanking both by the
target sequence given in column C. These transposons were then
transfected into CHO-K1 cells together with genes encoding
transposases whose SEQ ID NO is shown in column F, operably linked
to the CMV promoter. The amount of each DNA in each transfection is
shown in columns E (transposon) and H (transposase) of Table 1.
[0153] CHO-K1 cells were transfected and puromycin-selected as
described in Section 4.2.1. Fluorescence was measured at Ex/Em of
488/518 nm, and is a measure of expression of the ORF encoding
fluorescent reporter DasherGFP from stably integrated transposons,
fluorescence from 3 independent transfections is shown in Table 1
columns J-L.
[0154] Table 1 rows 3, 5, 10 and 15 show the results from three
different transposons transfected into CHO cells without any
co-transfected transposase. In each case there were few to no live
cells that survived the puromycin selection (column I), and no
fluorescence from the Dasher GFP (columns J, K and L), indicating
that the transposons had either not integrated, or not integrated
in a way that allowed subsequent expression of the genes encoded on
the transposons.
[0155] Table 1 rows 3 and 4 compares fluorescence obtained from a
transposon with ends taken from the looper moth Trichoplusia ni
piggyBac transposon (SEQ ID NO: 35 and 36), either transfected
alone (row 3) or co-transfected with a plasmid carrying a gene
encoding the hyperactive piggyBac transposase (SEQ ID NO 698)
operably linked to the CMV promoter (row 4). Co-transfection with
the transposase gene increased cell viability to give 100%
confluence, and the fluorescent signal increased from background to
.about.660 units.
[0156] Table 1 rows 5-14 compares fluorescence obtained from a
transposon with ends with SEQ ID NO: 1 and 11, either transfected
alone (row 5) or co-transfected with a gene encoding Xenopus
transposase SEQ ID NO: 49 alone (row 7) or fused to a heterologous
nuclear localization signal (row 6), or co-transfected with a gene
encoding Xenopus transposase SEQ ID NO: 48 alone (row 9) or fused
to a heterologous nuclear localization signal (row 8); or a
transposon with ends with SEQ ID NO: 3 and 12, either transfected
alone (row 10) or co-transfected with a gene encoding Xenopus
transposase SEQ ID NO: 49 alone (row 12) or fused to a heterologous
nuclear localization signal (row 11), or co-transfected with a gene
encoding Xenopus transposase SEQ ID NO: 48 alone (row 14) or fused
to a heterologous nuclear localization signal (row 13).
Co-transfection with either transposase fused to a nuclear
localization signal increased cell viability to give 100%
confluence, and the fluorescent signal increased from background to
.about.1,000 units. In the absence of the nuclear localization
signal viable cells and expression levels were less than 10% of the
values obtained with the transposases fused to heterologous nuclear
localization signals. Heterologous nuclear localization signals are
thus required for naturally occurring Xenopus transposases (for
example SEQ ID NOS: 48 and 49) to efficiently integrate transposons
into the nucleus of mammalian cells in a way that allows subsequent
expression of the genes encoded on the transposons.
[0157] The data in Table 1 shows that, when fused to a heterologous
nuclear localization signal, Xenopus transposases SEQ ID NO: 48 and
49 are active at transposing synthetic Xenopus transposons into the
genome of a mammalian cell. These transposon ends each contain an
ITR with the sequence 5'-CCYTTTBMCTGCCA-3' (SEQ ID NO: 19), where
the ITRs are found in the two ends in an inverted orientation
relative to each other. It also shows that the fusion of these
transposases to a heterologous nuclear localization signal are more
active in this assay than the hyperactive piggyBac transposase
derived from the looper moth Trichoplusia ni.
[0158] Table 1 rows 15-19 compares fluorescence obtained from a
transposon with ends with SEQ ID NO: 23 and 29, either transfected
alone (row 15) or co-transfected with a gene encoding Bombyx
transposase SEQ ID NO: 750 alone (row 17) or fused to a
heterologous nuclear localization signal (row 16), or
co-transfected with a gene encoding Bombyx transposase SEQ ID NO:
407 alone (row 19) or fused to a heterologous nuclear localization
signal (row 18). Co-transfection with transposase SEQ ID NO: 407,
whether or not it was fused to a nuclear localization signal
increased cell viability to give 100% confluence, and the
fluorescent signal increased from background to .about.1,000 units.
Co-transfection with transposase SEQ ID NO: 750, whether or not it
was fused to a nuclear localization signal resulted in viable cells
and expression levels were less than 1% of the values obtained with
the transposase SEQ ID NO: 407. Thus transposase SEQ ID NO: 407 is
active at transposing synthetic Bombyx transposons into the genome
of a mammalian cell.
6.1.2 Xenopus- and Bombyx-Derived Transposons
6.1.2.1 Bombyx Transposon Ends
[0159] Transposon ends of naturally occurring transposons were
modified by truncation or by changing the target sequences. These
transposon ends were then joined to the ends of a heterologous
polynucleotide to create synthetic transposons that can be
integrated into a target genome by a transposase. Table 2 shows the
configurations of 12 different synthetic transposons created by
joining the transposon end whose SEQ ID NO is given in column A to
one side of the reporter construct SEQ ID NO: 39, joining the
transposon end whose SEQ ID NO is given in column B to the other
side of the reporter construct and flanking both by the target
sequence given in column C. These transposons were then transfected
into CHO-K1 cells together with genes encoding transposases whose
SEQ ID NO is shown in column G, optionally fused to a heterologous
nuclear localization signal (as shown in column H) and operably
linked to the CMV promoter. The amount of each DNA in each
transfection is shown in columns F (transposon) and I (transposase)
of Table 2. Transfection and selection were as described in Section
4.2.1.
[0160] Cells were harvested by scraping and measured in a
fluorimetric plate reader, fluorescence from 3 independent
transfections is shown in Table 2 columns J-L. Fluorescence was
measured at Ex/Em of 488/518 nm, and is a measure of expression of
the ORF encoding fluorescent reporter DasherGFP from stably
integrated transposons.
[0161] Bombyx left and right transposon ends could both be
truncated from the proximal end (that is the end furthest from the
ITR) while retaining transposon function. Table 2 rows 2-9 show
that expression from heterologous polynucleotides inserted into the
CHO genome was enhanced by co-transfection with a construct
encoding Bombyx transposase SEQ ID NO: 407 wherein the heterologous
polynucleotides comprised a left transposon end of a target
sequence followed by SEQ ID NO: 23, 24 or 25, and a right
transposon end of SEQ ID NO: 29 or 31 followed by a target
sequence.
[0162] The test performed here shows that these ends comprise all
of the sequences necessary to create a Bombyx transposon that can
be integrated into the genome of a target cell. However, it has
previously been shown for the looper moth piggyBac transposon that
longer sequences are required for transformation of target genomes
than for excision of the transposon by the transposase, or for
inter-plasmid transposition, as described in Li et. al (2005)
Insect Mol. Biol. 14: 17-30. "piggyBac internal sequences are
necessary for efficient transformation of target genomes." and Li
et. al (2001) Mol Genet Genomics 266:190-8. "The minimum internal
and external sequence requirements for transposition of the
eukaryotic transformation vector piggyBac.". We infer that shorter
sequences of the Bombyx transposon will also be competent for
excision or for inter-plasmid transposition. Important sequences
for looper moth piggyBac transposon excision are the terminal
repeats and internal repeats in each end. The Bombyx transposon
comprises several internal repeats which probably perform analogous
functions. Bombyx left end SEQ ID NO: 25 comprises SEQ ID NO: 1103,
and an inverted copy of this SEQ ID NO: 1104; it also comprises SEQ
ID NO: 1105, and an inverted copy of this SEQ ID NO: 1106; it also
comprises two AT rich palindromes SEQ ID NO: 1107 and SEQ ID NO:
1108. Bombyx right end SEQ ID NO: 31 comprises two copies of the AT
rich sequence SEQ ID NO: 1110. Bombyx right end SEQ ID NO: 31 also
comprises a copy of SEQ ID NO: 1106, which is found repeated in
both orientations in left end SEQ ID NO: 25. Bombyx left end SEQ ID
NO: 25 and right end SEQ ID NO: 31 also each comprise a copy of SEQ
ID NO: 1109. A Bombyx transposon can comprise a left end comprising
1 or 2 or 3 or 4 or 5 or 6 or 7 sequences selected from SEQ ID NO:
1103-1110. A Bombyx transposon can comprise a right end comprising
1 or 2 or 3 sequences selected from SEQ ID NO: 1106 and
1109-1110.
[0163] We also found that we could change the 5'-TTAT-3' target
sequence flanking the Bombyx-based transposon to 5'-TTAA-3' and
still obtain a high transposase-dependent DasherGFP signal (compare
rows 3 and 11 in Table 2). Thus a Bombyx transposase is effective
at transposing transposons with different target sequences
including 5'-TTAT-3' and 5'-TTAA-3' target sequences.
6.1.2.2 Xenopus Transposon Ends
[0164] Tables 2 and 3 also show expression from Xenopus transposons
with truncated ends or modified target sequences. Both tables show
the configurations of synthetic transposons created by joining the
transposon end whose SEQ ID NO is given in column A to one side of
the reporter construct SEQ ID NO: 39, joining the transposon end
whose SEQ ID NO is given in column B to the other side of the
reporter construct and flanking both by the target sequence given
in column C. These transposons were then transfected into CHO-K1
cells together with genes encoding transposases whose SEQ ID NO is
shown in column G, optionally fused to a heterologous nuclear
localization signal (as shown in column H) and operably linked to
the CMV promoter. The amount of each DNA in each transfection is
shown in columns F (transposon) and I (transposase) of Tables 2 and
3. Transfection and selection were as described in Section
4.2.1.
[0165] Cells were harvested by scraping and measured in a
fluorimetric plate reader, fluorescence from 3 independent
transfections is shown in Tables 2 and 3 columns J-L. Fluorescence
was measured at Ex/Em of 488/518 nm, and is a measure of expression
of the ORF encoding fluorescent reporter DasherGFP from stably
integrated transposons.
[0166] Table 2 rows 13-20 and Table 3 rows 2-11 show that
expression from heterologous polynucleotides inserted into the CHO
genome was enhanced by co-transfection with a construct encoding
Xenopus transposase SEQ ID NO: 48 fused to a nuclear localization
sequence, wherein the heterologous polynucleotides comprised a left
transposon ends of a target sequence followed by SEQ ID NO: 1 or
3-7 and a right transposon end of SEQ ID NO: 11-13 or 15-16
followed by a target sequence.
[0167] The test performed here shows that these ends comprise all
of the sequences necessary to create a Xenopus transposon that can
be integrated into the genome of a target cell. However, it has
previously been shown for the looper moth piggyBac transposon that
longer sequences are required for transformation of target genomes
than for excision of the transposon by the transposase, or for
inter-plasmid transposition, as described in Li et. al (2005)
Insect Mol. Biol. 14: 17-30. "piggyBac internal sequences are
necessary for efficient transformation of target genomes." and Li
et. al (2001) Mol Genet Genomics 266:190-8. "The minimum internal
and external sequence requirements for transposition of the
eukaryotic transformation vector piggyBac.". We infer that shorter
sequences of the Xenopus transposon will also be competent for
excision or for inter-plasmid transposition.
[0168] We also found that we could change a 5'-TTAA-3' target
sequence flanking the Xenopus-based transposon to a 5'-TTAT-3'
target sequence and still obtain a high transposase-dependent
DasherGFP signal (compare rows 14 and 22 in Table 2). The ITRs for
these transposons were adjacent to the left target sequence,
sequence SEQ ID NO: 20 (5'-CCCTTTGCCTGCCA-3'), and adjacent to the
right target sequence, sequence SEQ ID NO: 21
(5'-TGGCAGTGAAAGGG-3').
[0169] Thus a Xenopus transposase is effective at transposing
transposons with different target sequences including 5'-TTAT-3'
and 5'-TTAA-3' target sequences.
[0170] We also found that we could change the 5'-TTAA-3' target
sequence flanking the Trichoplusia ni-based transposon to
5'-TTAT-3' and still obtain a high transposase-dependent DasherGFP
signal (compare rows 24 and 26 in Table 2). Thus the Trichoplusia
ni piggyBac transposase is effective at transposing transposons
with different target sequences including 5'-TTAT-3' and 5'-TTAA-3'
target sequences.
6.1.3 Transposon Integration by Transposase Provided as mRNA
6.1.3.1 Xenopus Transposase mRNA
[0171] A transposase may be provided as a protein, or as a
polynucleotide encoding the transposase; the encoding
polynucleotide may be expressible DNA or RNA. mRNA encoding Xenopus
transposase SEQ ID NO: 48 fused to a heterologous NLS was prepared
as described in Section 4.2.3.
[0172] Transposon ends were joined to the ends of a heterologous
polynucleotide to create synthetic transposons: the transposon ends
whose SEQ ID NOs are given in columns A and B of Table 5 were
joined to either side of reporter construct SEQ ID NO: 39, and
flanked by 5'-TTAA-3' target sequences. These transposons were
transfected into CHO-K1 cells together with polynucleotides
encoding transposases whose SEQ ID NO is shown in column E of Table
5. If the polynucleotide was DNA, the gene encoding the transposase
was operably linked to the promoter indicated in column G, and the
amount of transposase gene DNA per transfection is indicated in
column H. If the polynucleotide was provided as mRNA, the amount of
RNA per transfection is indicated in column I. The amount of each
transposon DNA in each transfection is shown in column D of Table
5. Transfection and selection was as described in Section
4.2.1.
[0173] Cells were harvested by scraping and measured in a
fluorimetric plate reader. Fluorescence, shown in columns J-L, was
measured at Ex/Em of 488/518 nm, and is a measure of expression of
the ORF encoding fluorescent reporter DasherGFP from stably
integrated transposons.
[0174] Table 5 shows that co-transfection of a transposon with
Xenopus transposon ends (each comprising SEQ ID NO: 19) together
with mRNA encoding a Xenopus transposase fused to a heterologous
NLS, resulted in up to 50.times. increases in expression relative
to the cells transfected with transposon alone, and comparable to
the expression enhancement obtained when the transposase was
provided encoded in DNA (compare rows 9-12 with rows 5-8 and row 4.
Also compare rows 18-21 with rows 14-17 and row 13). Thus a Xenopus
transposase may be provided as mRNA that can be translated in the
target cell.
6.1.3.2 Bombyx Transposase mRNA
[0175] A similar experiment to the one described in 6.1.3.1 was
performed with mRNA encoding Bombyx transposase 407 fused to a
heterologous NLS, also prepared by in vitro transcription as
described in Section 4.2.3.
[0176] Transposon ends were joined to the ends of a heterologous
polynucleotide to create synthetic transposons that can be
integrated into a target genome by a transposase. Table 6 shows the
configurations of 3 different synthetic transposons. Transposons
had a 5'-TTAA-3' target sequence followed by a transposon end whose
SEQ ID NO is given in column A, followed by the reporter construct
whose SEQ ID NO is given in column B, followed by the transposon
end whose SEQ ID NO is given in column C, followed by a 5'-TTAA-3'
target sequence.
[0177] These transposons were transfected into CHO-K1 cells
together with polynucleotides encoding transposases whose SEQ ID NO
is shown in column F of Table 6. If the polynucleotide was DNA, the
gene encoding the transposase was operably linked to the CMV
promoter, and the amount of transposase gene DNA per transfection
is indicated in column H. If the polynucleotide was provided as
mRNA, the amount of RNA per transfection is indicated in column I.
The amount of each transposon DNA in each transfection is shown in
column E of Table 6. Transfection and selection were as described
in Section 4.2.1. Cells were harvested by scraping and measured in
a fluorimetric plate reader. Fluorescence, shown in columns J-L,
was measured at Ex/Em of 488/518 nm, and is a measure of expression
of the open reading frame (ORF) encoding fluorescent reporter
DasherGFP from stably integrated transposons.
[0178] Table 6 shows that co-transfection of a transposon with
Bombyx transposon ends (each comprising SEQ ID NO: 33) together
with mRNA encoding a Bombyx transposase fused to a heterologous
NLS, resulted in up to 100.times. increases in expression relative
to the cells transfected with transposon alone, and comparable to
the expression enhancement obtained when the transposase was
provided encoded in DNA (compare rows 5-8 with rows 9-11 and row 4.
Also compare rows 13-16 with rows 17-19 and row 12). Thus a Bombyx
transposase may be provided as mRNA that can be translated in the
target cell.
6.1.3.3 Hyperactive Xenopus Transposase mRNA
[0179] It is advantageous to provide a transposase as expressible
RNA, since this avoids any possibility that the transposase gene
may be integrated into the target genome. Messenger RNA encoding
hyperactive Xenopus transposases, SEQ ID NO: 168, 189 and 175,
fused to a heterologous NLS were prepared by in vitro transcription
using T7 RNA polymerase as described in Section 4.2.3.
[0180] A transposon comprised 5'-TTAA-3' target sequences,
transposon end sequences SEQ ID Nos: 2 and 12, and a CMV enhancer
and CMV promoter operably linked to a gene encoding DasherGFP. The
transposon also comprised the murine phosphoglycerate kinase (PGK)
promoter, SEQ ID NO: 937, operably linked to a gene encoding
puromycin N-acetyl transferase.
[0181] The transposon was transfected into CHO-S suspension cells
together with polynucleotides encoding transposases whose SEQ ID NO
is shown in column C of Table 12. The amount of transposase mRNA
per transfection is indicated in column F. The amount of each
transposon DNA in each transfection is shown in column E of Table
12.
[0182] CHO-S cells were transfected as described in Section 4.2.2.
Puromycin (50 .mu.g/ml) selection was carried out for 10 days, and
cells were grown for 5 days post puromycin selection with two
passages and changes of media. Cells were harvested by pipetting
directly into a fluorimetric plate and measured in a fluorimetric
plate reader. Fluorescence was measured at Ex/Em of 488/518 nm, and
is a measure of expression of the ORF encoding fluorescent reporter
DasherGFP from stably integrated transposons.
[0183] Table 12 shows that there was no significant difference in
fluorescence between samples at 72 hours post-transfection.
However, after 10 days of puromycin treatment, fluorescence from
all transposons that had been co-transfected with a hyperactive
transposase mRNA was between 5 and 30-fold brighter than from
transposons co-transfected with mRNA encoding natural Xenopus
transposase SEQ ID NO: 48. After 5 days of recovery, all
hyperactive transposase co-transfections were still outperforming
SEQ ID NO: 48. Thus hyperactive transposases identified using a
functional screen in Saccharomyces cerevisiae lead to reduced
recovery times and increased expression from transposons in
mammalian cells, and hyperactive Xenopus transposases may be
provided as mRNA that can be translated in the target cell.
6.1.3.4 Hyperactive Xenopus and Bombyx Transposase mRNA
[0184] Messenger RNA encoding hyperactive Xenopus transposases SEQ
ID NO: 175 and 189, fused to a heterologous NLS, or Bombyx
transposases SEQ ID NO: 407 and 1098 were prepared by in vitro
transcription using T7 RNA polymerase as described in Section
4.2.3.
[0185] Transposons comprised transposon end sequences with SEQ ID
NOs given in columns B and C of Table 13. The transposons further
comprised a CMV enhancer and CMV promoter operably linked to a gene
encoding DasherGFP, and a gene encoding puromycin acetyl
transferase operably linked to a promoter with SEQ ID NO shown in
column F. Transposons (750 ng) were co-transfected with 250 ng mRNA
encoding a transposase with SEQ ID NO shown in column G, fused to a
heterologous nuclear localization signal.
[0186] CHO-S cells (from ATCC) were transfected as described in
Section 4.2.2. Puromycin (10 .mu.g/ml) selection was carried out
for 10 days, with a complete media change into fresh
puromycin-containing media after 5 days. After 10 days, cells were
transferred to fresh media containing 25 .mu.g/ml puromycin for 4
days. Cells were grown for 5 days post puromycin selection with two
passages and changes of media. Cells were harvested by pipetting
directly into a fluorimetric plate and measured in a fluorimetric
plate reader. Fluorescence was measured at Ex/Em of 488/518 nm, and
is a measure of expression of the ORF encoding fluorescent reporter
DasherGFP from stably integrated transposons, triplicate
measurements are shown in columns H-J of Table 13.
[0187] Table 13 shows that mRNAs encoding hyperactive Xenopus or
Bombyx transposases are active on their respective transposons.
6.2 Polynucleotide Constructs for Expression of Multiple
Proteins
6.2.1 Transposons with Dual Promoter or IRES Configurations (1)
[0188] Transposons and transposases may be used to efficiently
integrate polynucleotide constructs encoding two or more
polypeptides into the genome of a cell. Transposons are useful for
this purpose because a transposase will usually integrate all of
the DNA between the two transposon ends during the transposition
process. This means that sequence elements that are configured to
achieve a specific ratio of expression between the different
encoded genes are more likely to be preserved, than if random
fragments of the polynucleotide are inserted into the genome of the
target cell. It also means that all of the encoded genes will be
integrated at each integration event which is useful with larger
polynucleotide constructs.
[0189] Table 7 shows the configurations of a set of transposons
comprising 5'-TTAA-3' target sequences and transposon ends SEQ ID
NOS: 2 and 12. Transposons comprised genes encoding DasherGFP and
CayenneRFP, except for rows 1 and 2 in which only one fluorescent
protein was present (columns C and D). When both fluorescent
protein genes were present, the DasherGFP gene always occurred
first. All of the transposons also comprised a puromycin resistance
gene operably linked to the PGK promoter and transcribed in the
opposite direction to the genes for the fluorescent proteins. All
of the transposons also comprised a pair of HS4 insulators, one
adjacent to each transposon end. In some configurations (rows 8-23)
the two genes were each operably linked to separate promoters and
polyadenylation signals, in some configurations the genes were
operably linked to a single promoter preceding the first gene and a
single polyA signal following the second gene, where the two genes
were operably linked by an IRES sequence or a CHYSEL sequence. The
transposons comprised the SEQ ID NO given in column A between the
first and second open reading frames. Regulatory elements
associated with each gene are shown in columns E-L of Table 7. The
number 1 in the headers indicates promoters preceding or polyA
signals following the first gene, the number 2 in the headers
indicates promoters preceding or polyA signals following the second
gene. All of these sequences further comprised expression enhancing
sequence SEQ ID NO: 866 sequence preceding the last polyadenylation
signal. Transposons were transfected into CHO cells together with a
gene encoding a transposase (SEQ ID NO: 48) fused to a nuclear
localization signal, operably linked to the CMV promoter. CHO cells
were transfected and selected as described in Section 4.2.1.
Fluorescence represents expression of the ORFs encoding fluorescent
reporter DasherGFP from stably integrated transposons measured at
Ex/Em of 488/518 nm and CayenneRFP was measured at Ex/Em of 525/580
nm. Mean fluorescence from triplicate independent transfections are
shown in Table 7 columns M and N respectively.
[0190] Column O in Table 7 shows the ratio of red to green
fluorescence obtained for each transposon. Because the two
fluorescent proteins fluoresce with different intensities, this is
not a measurement of the ratios of concentration of the two
proteins. Row 3 shows the fluorescence obtained when the two
proteins are coupled by a CHYSEL sequence. This produces close to
equimolar amounts of the two proteins, and gives a red to green
fluorescence ratio of 0.21. Column P shows the red to green ratios
of column 0, normalized by dividing by 0.21, to obtain the ratio at
which the two proteins are expressed. Column Q shows the expression
of DasherGFP relative to the expression when DasherGFP is the only
encoded fluorescent protein.
[0191] Coupling the translation of two open reading frames through
IRES SEQ ID NOS: 1062-1064 produced very high levels of expression
of the first encoded protein (at least 70% of the amount of protein
obtained from a monocistronic construct, compare row 2 with rows
4-7). These constructs also produced the second encoded protein at
between 0.33 and 0.48 the levels of the first. Ratios or 1:0.33 to
1:0.48 are very good for production of light and heavy chains of
antibodies respectively. Thus IRES SEQ ID NOS: 1062-1064 are
preferred components of polynucleotide constructs for the efficient
production of antibodies.
[0192] Another way to obtain expression of two genes from one
polynucleotide is to operably link each gene to a separate
promoter. Rows 8-23 of Table 7 show a variety of different
configurations for the regulatory elements associated with the
second open reading frame. In general, the expression of the first
encoded protein was less in these constructs than in the
IRES-coupled constructs (column Q). However, a greater range of
expressions of the second encoded gene were obtained (column P).
Expression of one gene and often both genes was substantially
increased if an HS4 core insulator (SEQ ID NO: 865) was interposed
between the polyadenylation sequence operably linked to the first
open reading frame, and the promoter operably linked to the second
open reading frame (compare rows 14 and 15, rows 16 and 17, rows 18
and 19, rows 20 and 21, rows 22 and 23, and rows 12 and 13). The
placement of an insulator sequence such as the HS4 core insulator
sequence between a polyadenylation sequence operably linked to a
first open reading frame, and a promoter operably linked to a
second open reading frame, is thus a preferred configuration for
expressing two genes from the same polynucleotide.
[0193] Advantageous vector configurations for the expression of two
polypeptides include those in which a gene encoding a first
polypeptide is operably linked to control elements including an EF1
intron from one species, and a gene encoding a second polypeptide
is operably linked to control elements including an EF1 intron from
a second species. Advantageous vector configurations for the
expression of two polypeptides include those in which a gene
encoding a first polypeptide is operably linked to control elements
including an EF1 promoter from one species, and a gene encoding a
second polypeptide is operably linked to control elements including
an EF1 promoter from a second species. Advantageous vector
configurations for the expression of two polypeptides include those
in which a gene encoding a first polypeptide is operably linked to
control elements including a human CMV promoter, and a gene
encoding a second polypeptide is operably linked to control
elements including a murine CMV promoter.
[0194] Advantageous vector configurations for the expression of two
polypeptides include those in which a gene encoding a first
polypeptide is operably linked to control elements including a
sequence that is at least 95% identical to a sequence selected from
SEQ ID NO: 1015, 1019, 1022, 1026, 1027, 1028, 1029 or 1099.
6.2.2 Transposons with Dual Promoter or IRES Configurations (2)
[0195] Configurations of a set of transposons comprising genes
encoding DasherGFP and/or CayenneRFP are indicated in Table 8
(columns D and E). When both fluorescent protein genes were
present, the DasherGFP gene always occurred first. All of the
transposons also comprised a puromycin resistance gene operably
linked to the PGK promoter and transcribed in the opposite
direction to the genes for the fluorescent proteins. Two of the
transposons (Transposons 188209 and 188219, column B) also
comprised a pair of HS4 insulators, one adjacent to each transposon
end. In some configurations the two genes were each operably linked
to separate promoters and polyadenylation signals, in some
configurations the genes were operably linked to a single promoter
preceding the first gene and a single polyA signal following the
second gene, where the two genes are operably linked by an IRES
sequence or a CHYSEL sequence. The number 1 indicates promoters
preceding or polyA signals following the first gene, the number 2
indicates promoters preceding or polyA signals following the second
gene. All of these sequences further comprised expression enhancing
sequence SEQ ID NO: 866 preceding the last polyadenylation signal.
Transposons were transfected into CHO cells plus or minus a gene
encoding a transposase (SEQ ID NO: 48) fused to a nuclear
localization signal, operably linked to the CMV promoter (column
O).
[0196] CHO-K1 cells were transfected and selected as described in
Section 4.2.1. Fluorescence represents expression of the ORFs
encoding fluorescent reporter DasherGFP from stably integrated
transposons measured at Ex/Em of 488/518 nm and CayenneRFP was
measured at Ex/Em of 525/580 nm (Table 8 columns P-U)
[0197] Co-transfection of transposons with the vector encoding the
transposase increased expression of both proteins encoded by the
transposon between 4-fold and nearly 20-fold relative to
transfections with the transposon alone. Expression of two
polypeptides from a transposon are improved by the activity of a
transposase. The best expressing transposons comprised flanking HS4
insulators, and expression of the two polypeptides coupled by an
IRES. These are thus preferred configurations for transposons for
expression of two polypeptides.
6.3 Modified Transposases
6.3.1 Hyperactive Xenopus Transposases
6.3.1.1 Identification of Hyperactive Xenopus Transposases
[0198] To identify Xenopus transposase mutations that led to either
increased transposition activity, or increased excision activity,
relative to the naturally occurring transposase sequence SEQ ID NO:
48, we first created libraries that together contained all possible
single amino acid changes from SEQ ID NO: 48, fused to a
heterologous nuclear localization sequence. To do this, a gene
encoding Xenopus transposase fused to a heterologous nuclear
localization sequence (SEQ ID NO: 50) was amplified by PCR, using
degenerate primers to incorporate all possible amino acids at a
single position. This "site-saturation" library was cloned into a
vector comprising a leucine selectable marker; genes encoding the
transposase mutants were operably linked to the Saccharomyces
cerevisiae Gal-1 promoter. One library was created for each of the
589 amino acids in the transposase. Each library was sequenced
across the mutagenized position to ensure that no unintended
mutations had been introduced, and that the targeted codon was
indeed mutated.
[0199] The cloned libraries were then pooled, each pool comprising
libraries for six adjacent amino acid positions, i.e. amino acids
1-6, 7-12, 13-18 etc. Each library pool was then transformed into
Saccharomyces cerevisiae cells that were carrying a genomically
integrated Xenopus transposon selection cassette, and plated on
minimal complete media lacking leucine to select for transformants
carrying the leucine selectable marker.
[0200] The Xenopus transposon selection cassette (SEQ ID NO: 44)
comprised the Saccharomyces cerevisiae URA3 promoter followed by a
gene encoding the first part of the Saccharomyces cerevisiae URA3
protein. The URA3 protein was interrupted at a TTAA sequence within
its coding region by a transposon insert comprising Xenopus left
transposon end SEQ ID NO: 2, the TEF promoter from Ashbya gossypii,
an open reading frame encoding the Saccharomyces cerevisiae TRP1
protein, the TEF terminator from Ashbya gossypii and Xenopus right
transposon end SEQ ID NO: 12. On the other side of the transposon
insert was a DNA sequence encoding the remainder of the
Saccharomyces cerevisiae URA3 protein, followed by the
Saccharomyces cerevisiae URA3 terminator. The transposon was
inserted such that the TTAA sequence was present at both ends, and
removal of the transposon to leave a single copy of this TTAA would
result in a complete and functional gene encoding the Saccharomyces
cerevisiae URA3 protein.
[0201] Two days after plating, transformed Saccharomyces cerevisiae
cells were harvested by adding 5 ml sterile water to each plate,
and gently scraping to resuspend the cells. Cells were combined
into pools representing 60 adjacent amino acids, i.e. 1-60, 61-120,
121-180 etc. The A.sub.600 of each pool was measured, and used to
estimate the concentration of live cells. Plasmid DNA was prepared
from a portion of each pool for sequencing. This was to determine
the frequency of each amino acid change in the naive (unselected)
library. The cells were then selected under 3 different
regimes.
[0202] Selection 1: 2.times.10.sup.8 cells from each pool were
transferred to minimal media minus leucine containing 2% galactose,
and grown for 4 hours at 30.degree. C. Cells were then plated onto
minimal complete media lacking uracil, tryptophan and leucine. Two
days after plating, transformed Saccharomyces cerevisiae cells were
harvested by adding 5 ml sterile water to each plate, and gently
scraping to resuspend the cells. Plasmid DNA was prepared to
determine the frequency of each amino acid change in the selected
library.
[0203] Selection 2: 2.times.10.sup.8 cells from each pool were
transferred to minimal media minus leucine containing 2% galactose,
and grown for 20 hours at 30.degree. C. Cells were then plated onto
minimal complete media lacking uracil, tryptophan and leucine. Two
days after plating, transformed Saccharomyces cerevisiae cells were
harvested by adding 5 ml sterile water to each plate, and gently
scraping to resuspend the cells. Plasmid DNA was prepared to
determine the frequency of each amino acid change in the selected
library.
[0204] Selection 3: 2.times.10.sup.8 cells from each pool were
transferred to minimal media minus leucine containing 2% galactose,
and grown for 20 hours at 30.degree. C. Cells were then plated onto
minimal complete media lacking uracil, and leucine, and containing
0.5 g/L 5-fluoroanthranilic acid. Five days after plating,
transformed Saccharomyces cerevisiae cells were harvested by adding
5 ml sterile water to each plate, and gently scraping to resuspend
the cells. Plasmid DNA was prepared to determine the frequency of
each amino acid change in the selected library.
[0205] Selections 1 and 2 identified cells in which the transposase
had precisely excised the transposon from the uracil gene (so the
cells are URA+) and re-integrated into another site in the genome
(so the cells were TRP+). Selection 3 identified cells in which the
transposase had precisely excised the transposon from the uracil
gene (so the cells were URA+) but not re-integrated into another
site in the genome (so the cells were TRP- and resistant to
5-fluoroanthranilic acid).
[0206] The mutated transposase genes from the naive library and
each of the selected libraries were sequenced using an Illumina
HiSeq. Mutation frequencies from the naive library were compared
with the frequencies in the selected libraries. Mutations that were
more highly represented in libraries selected under conditions 1 or
2, compared with the naive library, were those that increase the
transpositional activity of the transposase. Mutations that were
more highly represented in the library selected under condition 3,
compared with the naive library, were those that increase the
excision activity of the transposase. Operability of a Xenopus
transposase can be shown by the ability of the transposase, when
fused to a heterologous NLS, to excise the transposon from within
SEQ ID NO: 44, and, except in the case of an integration-deficient
transposase, to integrate the transposon into the genomic DNA of a
target cell.
[0207] Table 4 shows the amino acid substitutions that were
represented at least 2 times more frequently in the selected
library than in the naive library. The data was processed as
follows. Considering each amino acid change independently, any
amino acid substitution that occurred in the naive library less
than once for each 200 substitutions observed at that position, was
discarded from further consideration. Any amino acid substitution
that was observed fewer than 100 times in the selected library was
discarded from further consideration. The frequency of each
substitution in each selected library was then calculated relative
to the frequency that the substitution occurred in the naive
library. Substitutions that occurred at least twice as frequently
in a library selected for transposition, compared with their
frequency in the naive library are shown in Table 4 column C.
Substitutions that occurred at least twice as frequently in a
library selected for excision, compared with their frequency in the
naive library are shown in Table 4 column D.
[0208] Some of the amino acid substitutions shown in Table 4 were
selected for incorporation into variant Xenopus transposases to
create hyperactive variants. Initially, a set of 95 variants of
transposase SEQ ID NO: 48 were created by selecting 57 of the
substitutions shown in Table 4, and incorporating 3 of these into
each of 95 variants, such that the number of possible pairs is
maximized and each substitution occurs 5 times in the set of
variants. The transposases were cloned into a vector comprising a
leucine selectable marker, so that the transposase variants were
operably linked to the Saccharomyces cerevisiae Gal-1 promoter.
Each of these variants was then individually transformed into a
Saccharomyces cerevisiae strain carrying a chromosomally integrated
copy of SEQ ID NO: 44, as described above. The variants were
induced with galactose, grown for 4 hours, then aliquots were
plated (a) on media lacking leucine, uracil and tryptophan (to
count integration), (b) on media lacking leucine and uracil (to
count excision) and (c) on media lacking leucine (to count total
live cells). Two days later, colonies were counted to determine
transposition (=number of cells on -leu-ura-trp media divided by
number of cells on -leu media) and excision (=number of cells on
-leu-ura media divided by number of cells on -leu media)
frequencies.
[0209] Transposition frequencies were modelled as described in U.S.
Pat. No. 8,635,029, and mean values and standard deviations for the
regression weights were calculated for each substitution.
Subsequent sets of variants were designed incorporating more than 3
substitutions relative to the sequence of SEQ ID NO: 48. These
variants combined two or more substitutions with regression weights
greater than one standard deviation above zero. The variants
optionally also comprised one or more substitution selected from
column C or D in Table 4. New variants were tested as described
above to measure transposition and/or excision frequencies for the
new variant transposases. Regression weights and standard
deviations for substitutions with a positive effect on
transposition activity are shown in Table 11 columns D and E.
Transposition frequencies for some hyperactive Xenopus transposases
are shown in Table 14 columns A and B. Frequencies were measured
for excision of the transposon from reporter SEQ ID NO: 44 and
integration of that transposon into the Saccharomyces cerevisiae
genome, and are expressed relative to the transposition frequencies
measured for the naturally occurring sequence SEQ ID NO: 48 under
identical conditions.
[0210] Transposases with SEQ ID NOS: 51 and 403-406 were found to
have excision frequencies that were at least 10-fold higher than
their integration frequencies. Transposases in which the amino acid
at position 218 was changed from Asn to either Asp or Glu also
showed much higher excision than integration frequencies. These
integration-deficient transposases are thus useful for removing
integrated transposons from a host genome.
6.3.1.2 Hyperactive Xenopus Transposases in Mammalian Cells
[0211] The ability of several hyperactive Xenopus transposases to
integrate three different transposon configurations into the CHO
genome was tested. Transposon configurations are shown in Table
16.
[0212] Transposons (750 ng) were co-transfected with
polynucleotides encoding Xenopus transposases fused to a
heterologous nuclear localization signal. Transposon and
transposase nucleic acids were transfected into 1 ml of
suspension-adapted CHO cells. Cells were grown for 72 hours
post-transfection and then diluted to 250,000 cells per ml in 40
.mu.g/ml puromycin for 8 days. The puromycin was removed and cells
were grown for a further 7 days. Expression of Dasher GFP was
measured in a plate fluorimeter (excitation at 485 nm and emission
measured at 515 nm).
[0213] This selection is highly stringent: the puromycin acetyl
transferase gene is operably linked with a weak promoter, and the
cells were diluted to low levels into high levels of puromycin. In
all cases under these stringent conditions, the absence of
transposase (Table 16 rows 3, 6 and 9) or co-transfection of
Xenopus transposons with naturally occurring Xenopus transposase
SEQ ID NO: 48 (Table 16 rows 1, 4 and 7), resulted in essentially
complete cell death and very low levels of DasherGFP expression. In
contrast co-transfection of Xenopus transposons with hyperactive
Xenopus transposases SEQ ID NOs: 57, 58 and 61 resulted in pools
with high levels of DasherGFP expression (Table 16 rows 2, 5 and
8). It is thus advantageous to co-transfect mammalian cells with
Xenopus transposons and hyperactive Xenopus transposases, including
SEQ ID NOS: 57, 58 and 61.
6.3.2 Hyperactive Bombyx Transposases
6.3.2.1 Identification of Hyperactive Bombyx Transposases
[0214] To identify Bombyx transposase mutations that led to either
increased transposition activity, or increased excision activity,
relative to naturally occurring sequence SEQ ID NO: 407, we first
created libraries that together contained all possible single amino
acid changes from SEQ ID NO: 407, fused to a heterologous nuclear
localization sequence. To do this, a gene encoding Bombyx
transposase fused to a heterologous nuclear localization sequence
(SEQ ID NO: 408) was mutagenized, cloned and sequenced as described
for the Xenopus transposase in Section 6.3.2.1.
[0215] The cloned libraries were pooled and transformed into
Saccharomyces cerevisiae cells which were carrying a genomically
integrated Bombyx transposon selection cassette as described for
the Xenopus transposase in Section 6.3.1.
[0216] The Bombyx transposon selection cassette (SEQ ID NO: 47) was
as described for the Xenopus cassette SEQ ID NO: 44 in Section
6.3.1, except that the Xenopus transposon ends SEQ ID NO 2 and 12
were replaced by Bombyx transposon end sequences SEQ ID NO 22 and
30 respectively. Operability of a Bombyx transposase can be shown
by the ability of the transposase, when fused to a heterologous
NLS, to excise the transposon from within SEQ ID NO: 47, and,
except in the case of an integration-deficient transposase, to
integrate the transposon into the genomic DNA of a target cell.
[0217] The Bombyx transposase mutant libraries were selected,
sequenced and processed, as described for the Xenopus transposase
libraries in section 6.3.1. Substitutions that occurred at least
twice as frequently in a Bombyx library selected for transposition,
compared with their frequency in the naive library are shown in
Table 4 column G. Substitutions that occurred at least twice as
frequently in a Bombyx library selected for excision, compared with
their frequency in the naive library are shown in Table 4 column
H.
[0218] Some of the amino acid substitutions shown in Table 4 were
selected for incorporation into Bombyx transposase SEQ ID NO: 407
to create hyperactive variants, as described for the Xenopus
transposase in Section 6.3.1 but using a Saccharomyces cerevisiae
strain carrying a chromosomally integrated copy of SEQ ID NO: 47 in
place of SEQ ID NO: 44.
[0219] Subsequent sets of variants were designed incorporating more
than 3 substitutions relative to the sequence of SEQ ID NO: 407.
These variants combined two or more substitutions with regression
weights greater than one standard deviation above zero. The
variants optionally also comprised one or more substitution
selected from column G or H in Table 4. New variants were tested as
described above to measure transposition and/or excision
frequencies for the new variant transposases. The regression
weights and standard deviations for substitutions with a positive
effect on transposition activity are shown in Table 11 columns I
and J. Transposition frequencies for different hyperactive Bombyx
transposases are shown in Table 14 columns C and D. Frequencies
were measured for excision of the transposon from reporter SEQ ID
NO: 47 and integration of that transposon into the Saccharomyces
cerevisiae genome, and are expressed relative to the frequencies
for the naturally occurring sequence SEQ ID NO: 407 under identical
conditions.
6.3.2.2 Hyperactive Bombyx Transposases in Mammalian Cells
[0220] The ability of several hyperactive Bombyx transposases to
integrate four different transposon configurations into the CHO
genome was tested. Transposon configurations are shown in Table 15.
Transposon 194094 comprised a PGK promoter (SEQ ID NO: 937)
operably linked to a puromycin acetyl transferase gene and a CMV
promoter operably linked to a gene encoding Dasher GFP. Transposon
240671 was the same as 194094, except that the transposon end
sequences were different, as shown in Table 15. Transposon 246143
was the same as 240671, except that the PGK promoter was replaced
with the HSV-TK promoter SEQ ID NO: 942. Transposon 246170 was
similar to 246143, but it had the EF1a promoter operably linked to
the gene encoding Dasher GFP, it is also flanked by insulator
sequences (HS4 insulator SEQ ID NO: 864 on one side and D4Z4
insulator SEQ ID NO: 860 on the other.
[0221] Transposons (750 ng) were co-transfected with
polynucleotides encoding a transposase with SEQ ID NO shown in
column K, fused to a heterologous nuclear localization signal.
Transposon and transposase nucleic acids were transfected into 1 ml
of suspension-adapted CHO cells. Cells were grown for 72 hours
post-transfection and then diluted to 1,000,000 cells per ml in 40
.mu.g/ml puromycin for 7 days. The puromycin was removed and cells
were grown for a further 7 days. Expression of Dasher GFP was
measured in a plate fluorimeter (excitation at 485 nm and emission
measured at 515 nm) (Table 15 columns O-Q). An estimate of live
cell numbers was made by measuring absorbance at 600 nm (A.sub.600)
(Table 15 columns L-N).
[0222] In all cases, the absence of transposase resulted in very
low levels of DasherGFP expression, and very low A.sub.600
indicating a lack of expression of puromycin acetyl transferase and
cell survival (rows 8, 16, 24 and 32). All transposases resulted in
comparable levels of cell survival for cells co-transfected with
transposons 194094 and 240671 (compare Table 15 columns L, M and N
for rows 1-7 and 9-15). However, the hyperactive transposases
resulted in significantly increased levels of DasherGFP expression
(compare Table 15 columns 0, P and Q for rows 1-7 and 9-15). It is
thus advantageous to co-transfect mammalian cells with Bombyx
transposons and hyperactive Bombyx transposases, including SEQ ID
NOS: 1098, 412, 457 and 415-417.
[0223] Cells transfected with transposon 246143 all died under the
selection conditions used, regardless of which transposase was
co-transfected (Table 15 columns L, M, N, O, P and Q for rows
17-24). However, cells transfected with transposon 246170 and
co-transfected with hyperactive Bombyx transposases, SEQ ID NOS:
1098, 412 and 415-417, all resulted in cells with Dasher GFP
fluorescence. No cells survived when this transposon was
co-transfected with the naturally occurring Bombyx transposase (SEQ
ID NO: 407). Hyperactive Bombyx transposases, SEQ ID NOS: 412 and
415 were particularly advantageous in combination with this
transposon configuration.
[0224] Transposon configuration, selection stringency and
transposase activity are interdependent in determining the
expression level that results from the subsequently integrated
transposon. The promoter that is operably linked to an expression
polypeptide (in this example DasherGFP) can also modify the
strength of the promoter that is operably linked to the selectable
marker and. As described in Section 5.2.10, a strong promoter
operably linked to the resistance marker (as in transposon 240671)
will provide the least stringent selection, while a weak promoter
operably linked to the resistance marker (as in transposon 246143)
will provide a more stringent selection, particularly in
combination with an interfering promoter operably linked with the
expression polypeptide.
[0225] The benefit of a more stringent selection coupled with a
hyperactive transposase is shown here. Hyperactive Bombyx
transposase SEQ ID NOS: 412 and 415 each produced a pool of cells
with substantially higher expression of the expression polypeptide
from transposon 246170 than was achieved from transposons with the
stronger promoter associated with the selectable marker (compare
row 9 column O with row 25 column O and row 26 column Q).
Furthermore, the productivity of the cells from the more
stringently selected transposon (expression divided by number of
live cells, which is approximately proportional to fluorescence
divided by A.sub.600) is about 10-fold higher than for the less
stringently selected transposon.
[0226] Although the relative integration frequencies of hyperactive
transposases shown in Table 14 give a quantitative comparison of
transposase activity, increased transposase activity alone is not
sufficient to guarantee increase expression resulting from
transposons integrated into a target cell genome. As shown in this
example, the transposon configuration and the selection stringency
are both factors that influence expression of an expression
polynucleotide. In particular the gene encoding the selectable
marker, the promoter (and other regulatory elements) operably
linked to the selectable marker, the promoter operably linked to
the gene encoding the expression polypeptide and any insulator
elements present are important determinants of expression from a
gene transfer polynucleotide. Particularly advantageous gene
transfer polynucleotides comprise a sequence that is at least 95%
identical to a sequence selected from SEQ ID NOS: 751-819.
Preferably these sequences are within a transposon.
[0227] The data shown in Table 15 shows only the average
fluorescence within a pool of cells. These pools were derived from
many independently transfected cells. Each of these will give rise
to a different transposon integration pattern (number of
transposons integrated and position of each of these transposons
within the target cell genome). Individual lines can be isolated
from a pool of cells like this, and some of these often have
substantially higher productivities than the pool.
6.4 IRES Elements
6.4.1 Expression Levels of Two Polypeptides Using IRES Elements in
HEK293 and Cho Cells
[0228] A gene transfer system comprising genes encoding two
polypeptides may operably link both polypeptides to the same
promoter, for example using an IRES.
[0229] Table 9 shows the expression levels observed in HEK and CHO
cells for two different polypeptides (in this case two different
fluorescent proteins, DasherGFP and CayenneRFP) encoded on a single
gene transfer polynucleotide. The genes for the two different
proteins were operably linked to a single enhancer, promoter,
polyadenylation signal and optionally an intron. Expression of the
two genes was operably linked by an IRES element, as indicated in
column A, with the order of elements being
DasherGFP-IRES-CayenneRFP.
[0230] HEK 293a cells (from ATCC) were grown in EMEM (from
ATCC)+10% FBS (from ATCC)+1% Penicillin-streptomycin (from ATCC) at
37.degree. C., 5% CO.sub.2 to 80% confluence, 1E+05 cells were
plated in 24-well tissue culture plates and incubated at 37.degree.
C., 5% CO.sub.2 for 24 hours prior to transfection, transfections
were set up in triplicates. Each transfection used 0.5 .mu.g DNA
with Lipofectamine 2000 as per manufacturer's protocol. Cells were
harvested 72 hours post transfection. CHO-K1 cells (from ATCC) were
grown in F12-K (from ATCC)+10% FBS (from ATCC)+1%
Penicillin-streptomycin (from ATCC) at 37.degree. C., 5% CO.sub.2
to 80% confluence. 5E+05 cells were plated in 24-well tissue
culture plates and incubated at 37.degree. C., 5% CO.sub.2 for 24
hours prior to transfection, transfections were set up in
triplicates. Each transfection used 0.5 .mu.g DNA with
Lipofectamine 2000 as per manufacturer's protocol. Cells were
harvested 72 hours post transfection. Fluorescence of the two ORFs
encoding fluorescent reporters DasherGFP and CayenneRFP was
measured at Ex/Em of 488/518 nm for DasherGFP and Ex/Em of 525/580
nm for CayenneRFP.
[0231] A gene transfer polynucleotide comprising the two proteins
translationally coupled by a CHYSEL sequence expresses the two
proteins at an equimolar ratio and was used to normalize for
different fluorescent intensities of the proteins. Table 9 shows
that different IRES elements can be used to obtain different ratios
of expression between two different polynucleotides. The use of
IRES elements is particularly advantageous for expression of
polypeptides when the ratio of expression is important at the level
of individual cells, for example in the expression of antibodies
where the light chain may perform a chaperonin function for the
heavy chain. It is sometimes advantageous to express as great a
ratio as possible between two polypeptides, for example in the case
when one polypeptide is a selectable marker.
[0232] We have identified IRES elements that show different levels
of activity as seen from the varying expression levels for the two
open reading frames (ORFs) linked by an IRES element shown in Table
9. A choice of IRES elements with varying activities allows the
appropriate IRES element to be used for controlling the relative
expression levels of two ORFs. We have shown use of one IRES
element linking two transcripts operably linked to one promoter.
Use of two or more IRES elements linking three or more ORFs is
expressly contemplated and is another aspect of the invention.
Expression constructs with two or more IRES elements selected such
that expression levels of two or more ORFs is selectively modulated
is expressly contemplated and is an important aspect of the
invention. The identified IRES elements of the invention work well
in both transient and stable integration vectors in the two cell
lines tested, Human embryonic kidney (HEK293) cells and Chinese
hamster ovary (CHO) cells. Preferred embodiments of a gene transfer
polynucleotide include all IRES elements shown in Table 9.
6.5 Transposase Activity in Yeast
6.5.1 Transposons in Pichia pastoris
[0233] To integrate a polynucleotide into the genome of Pichia
pastoris, it is generally necessary to linearize a gene transfer
construct prior to transformation. It is advantageous if the ends
of the linear gene transfer construct are homologous to neighboring
sequences in the Pichia pastoris genome, so that the construct may
be integrated into the chromosome by homologous recombination. Such
gene transfer constructs generally comprise a gene encoding a
selectable marker (for example resistance to zeocin (e.g. SEQ ID
NO: 702), nourseothricin (e.g. SEQ ID NO: 701) or geneticin (e.g.
SEQ ID NO: 706). High levels of expression may be obtained by
exposing cells to high levels of the corresponding selection agent,
which results in amplification of the gene. The amplification is
usually achieved by tandem duplication of the gene, which is an
inherently unstable arrangement. Because transposons integrate
almost randomly throughout the target genome, they offer the
advantage of high expression resulting from multiple inserted
copies, while improving stability because the copies are
distributed throughout the genome.
[0234] Three transposons were constructed for modifying the genome
of Pichia pastoris, and comprised transposon ends SEQ ID NOs: 2 and
12 flanking a heterologous polynucleotide. The heterologous
polynucleotide comprised an AOX promoter (SEQ ID NO: 953) operably
linked to a gene encoding Dasher GFP (SEQ ID NO: 42), and an ILV5
promoter (SEQ ID NO: 955) operably linked to a gene encoding zeocin
resistance (SEQ ID NO: 702). One of these transposons (251587) was
carried on a plasmid that comprised the GAP promoter SEQ ID NO: 949
operably linked to a gene encoding Xenopus transposase SEQ ID NO:
118; a second transposon (251588) was carried on a plasmid that
comprised the TEF promoter SEQ ID NO: 954 operably linked to a gene
encoding Xenopus transposase SEQ ID NO: 118; a third transposon
(251589) was carried on a plasmid with no transposase. All
transposases were part of the non-transposable portion of the
plasmid.
[0235] The three transposons were transformed as supercoiled
circular DNA into competent Pichia pastoris cells by
electroporation (using a Bio-Rad E. coli Pulser in cuvettes with a
0.2 cm gap, and 1.5 kV). In addition, transposon 251589 (whose
plasmid lacked a transposase entirely) was electroporated after
linearization with PmeI, which cuts within the AOX promoter. After
electroporation the cells were grown in non-selective media (900
.mu.l YPD broth plus 1M sorbitol) for 5 or 24 hours at 30.degree.
C., before 100 .mu.l culture was plated onto 200 .mu.g/ml zeocin
and plates incubated at 30.degree. C. for 48 hours. The number of
zeocin resistant colonies on each plate were counted, and are shown
in Table 10 (columns E and F).
[0236] Without linearization, very few colonies were formed in the
absence of a transposase (rows 9-11). By contrast, linearization
prior to electroporation resulted in approximately 1,000 colonies
from 100 .mu.l culture (row 12). Similarly, the expression
ofXenopus transposase SEQ ID NO: 118, either transcribed from the
GAP promoter (rows 3-5) or the TEF promoter (rows 6-8) resulted in
tens to hundreds of colonies. Xenopus transposons and transposases
are thus useful for integrating gene transfer constructs into the
genome of the yeast Pichia pastoris.
BRIEF DESCRIPTION OF TABLES
[0237] Table 1. Integration of Transposons Catalyzed by Modified
Transposases with or without Heterologous Nuclear Localization
Signals.
[0238] Transposons and transposases were transfected into CHO-K1
cells and selected as described in Example 6.1.1. Fluorescence was
measured by scraping cells and placing in a fluorimeter.
Fluorescent readings obtained in independent triplicate
transfections are shown in columns J-L. Columns A, B and F refer to
SEQ ID NOs.
Table 2. Integration of Transposons with Modified Transposon
Ends.
[0239] Transposons and transposases were transfected into CHO-K1
cells and selected as described in Example 6.1.2.1. Fluorescence
was measured by scraping cells and placing in a fluorimeter.
Fluorescent readings obtained in independent triplicate
transfections are shown in columns J-L. Columns A, B and G refer to
SEQ ID NOs.
Table 3. Integration of Transposons with Modified Transposon
Ends.
[0240] Transposons and transposases were transfected into CHO-K1
cells and selected as described in Example 6.1.2.2. Fluorescence
was measured by scraping cells and placing in a fluorimeter.
Fluorescent readings obtained in independent triplicate
transfections are shown in columns J-L. Columns A, B and G refer to
SEQ ID NOs.
Table 4. Substitutions in Transposases Associated with
Hyperactivity.
[0241] Mutant Xenopus and Bombyx transposases were produced,
selected and sequenced as described in Examples 6.3.1.1 and
6.3.2.1. Positions relative to Xenopus transposase SEQ ID NO: 48
are shown in column A; the naturally occurring amino acid is in
column B; substitutions that occurred at least twice as frequently
in a Xenopus library selected for transposition, compared with
their frequency in the naive library are shown in column C;
substitutions that occurred at least twice as frequently in a
Xenopus library selected for excision, compared with their
frequency in the naive library are shown in column D. Positions
relative to Bombyx transposase SEQ ID NO: 407 are shown in column
E; the naturally occurring amino acid is in column F; substitutions
that occurred at least twice as frequently in a Bombyx library
selected for transposition, compared with their frequency in the
naive library are shown in column G; substitutions that occurred at
least twice as frequently in a Bombyx library selected for
excision, compared with their frequency in the naive library are
shown in column H. Positions in the two transposases sharing a line
in the table do not correspond to a sequence alignment between the
two proteins.
Table 5. Integration of Transposons Using Transposase mRNA.
[0242] Transposons and transposases were transfected into CHO-K1
cells and selected as described in Example 6.1.3.1. Fluorescence
was measured by scraping cells and placing in a fluorimeter.
Fluorescent readings obtained in independent triplicate
transfections are shown in columns J-L. Columns A, B and E refer to
SEQ ID NOs.
Table 6. Integration of Transposons Using Transposase mRNA.
[0243] Transposons and transposases were transfected into CHO-K1
cells and selected as described in Example 6.1.3.3. Fluorescence
was measured by scraping cells and placing in a fluorimeter.
Fluorescent readings obtained in independent triplicate
transfections are shown in columns J-L. Columns A, B, C and F refer
to SEQ ID NOs.
Table 7. Integration of Xenopus-Derived Transposons for Expression
of Two Polypeptides.
[0244] Transposons comprised 5'-TTAA-3' target sequences,
transposon end sequences SEQ ID NO: 2 and SEQ ID NO: 12, and the
EF1a promoter and intron operably linked to a gene encoding
DasherGFP (rows 2-23) or CayenneRFP (row 1). For rows 3-7, vectors
further comprised a gene encoding CayenneRFP operably linked to the
expression control elements by a translational-coupling sequence
(SEQ ID NO given in column A). For rows 8-23, vectors further
comprised a gene encoding Cayenne RFP operably linked to a second
enhancer (column I), a second promoter (column J), a second intron
(column K) and a second polyadenylation signal (column L).
Optionally an insulator sequence was interposed between the first
polyadenylation signal and the second enhancer (column H). The
transposons comprised a sequence whose SEQ ID NO is given in column
A between the two ORFs. Transposons were transfected in triplicate
independent transfections into CHO cells together with a gene
encoding a transposase (SEQ ID NO. 48) fused to a heterologous
nuclear localization signal. Cells were selected and expression of
the fluorescent proteins measured (columns M and N show the
averages of 3 measurements for each fluorescent protein) as
described in Example 6.2.1.
Table 8. Integration of Xenopus-Derived Transposons for Expression
of Two Polypeptides.
[0245] Transposons comprised 5'-TTAA-3' target sequences,
transposon end sequences SEQ ID NO: 2 and 12, and an enhancer
(column F), promoter (column G) and intron (column H) operably
linked to a gene encoding DasherGFP. For rows 3-6, vectors further
comprised a gene encoding CayenneRFP operably linked to the
expression control elements by a translational-coupling sequence
(sequences identified in column A). For rows 7-18, vectors further
comprised a gene encoding Cayenne RFP operably linked to a second
enhancer (column K), a second promoter (column L) and a second
intron (column M). Polyadenylation signals were linked to the first
(column I) and second (column N) open reading frames. Optionally an
insulator sequence was interposed between the first polyadenylation
signal and the second enhancer (column J). Transposons comprised a
sequence whose SEQ ID NO is given in column A between the two ORFs.
Transposons were transfected into CHO cells, optionally (as
indicated in column O) together with a gene encoding a transposase
(SEQ ID NO: 48) fused to a heterologous nuclear localization
signal; cells were selected and expression of the fluorescent
proteins measured (columns P-U) as described in Example 6.2.2. Rows
1-2 and 19-20 show the transfection of constructs encoding only GFP
(rows 1-2) or RFP (rows 19-20). Rows 21 and 22 shows the
co-transfection of the constructs shown in rows 1 and 19. Details
are given in Section 6.2.2.
Table 9. Expression from Gene Transfer Systems Comprising Genes
Encoding Two Polypeptides Linked by IRES Translational Coupling
Elements (5).
[0246] Gene transfer polynucleotide s comprised an enhancer),
promoter, intron and polyadenylation signal operably linked to a
gene encoding DasherGFP, an IRES element and a gene encoding
CayenneRFP. IRES element SEQ ID NOs are given in column A). Vectors
were transfected into HEK or CHO cells and expression of the
fluorescent proteins measured as described in Example 6.4.1. The
relative fluorescence of the two proteins is shown in column B
(HEK) or F (CHO). The relative expression level of the CayenneRFP
to DasherGFP was calculated by correcting for the relative
fluorescence levels of the two proteins (CayenneRFP only yields
0.3.times. the signal of DasherGFP for the same protein level).
This is the IRES efficiency shown in column C (HEK) or G (CHO). The
expression level of the DasherGFP in the IRES construct was
compared with the expression of DasherGFP from a construct lacking
an IRES and CayenneRFP and is shown as % GFP shown in column D
(HEK) or H (CHO). The number of independent experiments measuring
expression of each IRES in each system is shown in column E (HEK)
or I (CHO).
Table 10. Xenopus Transposons in Pichia pastoris
[0247] Three transposons (column B) were constructed for modifying
the genome of Pichia pastoris, and comprised 5'-TTAA-3' target
sequences and transposon end SEQ ID Nos: 2 and 12 flanking a
heterologous polynucleotide as described in Section 6.5.1. Plasmids
in rows 3-8 also comprised a promoter (whose SEQ ID NO is given in
column D) operably linked to a gene encoding Xenopus transposase
SEQ ID NO: 118 on a non-transposable portion of the plasmid.
Different amounts (column D) of the three transposons were
transformed into competent Pichia pastoris cells, grown and plated
as described in Section 6.5.1. The number of zeocin resistant
colonies on each plate were counted after the cells were grown in
non-selective media (900 .mu.l YPD broth plus 1M sorbitol) for 5
(column E) or 24 hours (column F) at 30.degree. C.
Table 11. Substitutions Conferring Hyperactivity on Xenopus
Transposase.
[0248] Transposase variants were created, transposition frequencies
were measured and the effect of amino acid substitutions on
transposition frequencies were modelled as described in Sections
6.3.1 and 6.3.2. Column A shows the position of substitutions in
the Xenopus transposase numbering from the beginning of SEQ ID NO:
48, column B shows the identity of that amino acid in SEQ ID NO:
48, column C shows the identity of an amino acid substitution that
confers hyperactivity on the transposase, column D shows the mean
regression weight of that substitution and column E shows the
standard deviation of the regression weight. Column F shows the
position of substitutions in the Bombyx transposase numbering from
the beginning of SEQ ID NO: 407, column G shows the identity of
that amino acid in SEQ ID NO: 407, column H shows the identity of
an amino acid substitution that confers hyperactivity on the
transposase, column I shows the mean regression weight of that
substitution and column J shows the standard deviation of the
regression weight.
Table 12. Hyperactive Xenopus Transposase Active as mRNA in CHO
Cells.
[0249] Transposons were co-transfected into CHO cells with either
plasmid DNA (row 2) or mRNA (rows 3-14) encoding a Xenopus
transposase (with SEQ ID NO shown in column C) fused to a
heterologous nuclear localization signal. Amounts of transposon and
transposase nucleic acid transfected into 1 ml of CHO cells are
shown in columns E and F. Transfection, growth and selection were
as described in Section 6.1.3.3. Expression of Dasher GFP was
measured in a plate fluorimeter. Row 15 shows a no DNA control.
Columns H and I show fluorescence from duplicate samples 3 days
post-transfection but before selection, columns J and K show
fluorescence from duplicate samples immediately following a 10-day
selection, columns L and M show fluorescence from duplicate samples
5 days post-selection.
Table 13. Hyperactive Xenopus and Bombyx Transposases Active as
mRNA in CHO Cells.
[0250] Transposons comprised transposon end sequences with SEQ ID
NOs given in columns B and C flanked by target sequences indicated
in column D. The transposons further comprised a CMV enhancer and
CMV promoter operably linked to a gene encoding DasherGFP, and a
gene encoding puromycin acetyl transferase operably linked to a
promoter with SEQ ID NO shown in column F. Transposons (750 ng)
were co-transfected with 250 ng mRNA encoding a transposase with
SEQ ID NO shown in column G, fused to a heterologous nuclear
localization signal. Transfection, growth and selection were as
described in Section 6.1.3.4. Expression of Dasher GFP was measured
in a plate fluorimeter. Row 16 shows a no DNA control. Columns H, I
and J show fluorescence from triplicate samples.
Table 14. Relative Transposition Frequencies for Hyperactive
Transposases.
[0251] Transposase variants were created and transposition
frequencies measured in Saccharomyces cerevisiae as described in
Section 6.3.1 and 6.3.2. Column A shows the SEQ ID NO of
hyperactive Xenopus transposases, column B shows the transposition
frequency of the hyperactive transposase in Saccharomyces
cerevisiae, relative to the frequency for the naturally occurring
sequence SEQ ID NO: 48 under identical conditions. Column C shows
the SEQ ID NO of hyperactive Bombyx transposases, column D shows
the transposition frequency of the hyperactive transposase in
Saccharomyces cerevisiae, relative to the frequency for the
naturally occurring sequence SEQ ID NO: 407 under identical
conditions.
Table 15. Hyperactive Bombyx Transposases Active in CHO Cells.
[0252] Transposons comprised transposon end sequences with SEQ ID
NOs given in columns B and C, flanked by target sequences given in
column D. The transposons further comprised a promoter (column F)
operably linked to a gene encoding DasherGFP, and a gene encoding
puromycin acetyl transferase operably linked to a promoter with SEQ
ID NO shown in column E. Transposons (750 ng) were co-transfected
with 250 ng mRNA (column I) or DNA (column J) encoding a
transposase with SEQ ID NO shown in column K, fused to a
heterologous nuclear localization signal. Columns L, M and N show
the absorbance at 600 nm for triplicate samples. Columns 0, P and Q
show the DasherGFP fluorescence from the corresponding samples.
Experimental details are as given in Section 6.3.2.2.
Table 16. Hyperactive Xenopus Transposases Active in CHO Cells.
[0253] Transposons comprised insulator sequences with SEQ ID NOs
shown in columns D and E, inside transposon end sequences SEQ ID
NO: 2 and 12, flanked by 5'-TTAA-3' target sequences. The
transposons further comprised a promoter (column C) operably linked
to a gene encoding DasherGFP, and a gene encoding puromycin acetyl
transferase operably linked to a promoter with SEQ ID NO shown in
column B. Transposons (750 ng) were co-transfected with 250 ng DNA
encoding a transposase with SEQ ID NO shown in column F, fused to a
heterologous nuclear localization signal. Columns G, H and I show
the DasherGFP fluorescence from triplicate independent
transfections of the corresponding samples. Experimental details
are as given in Section 6.3.1.2.
7. REFERENCES
[0254] All references cited herein are incorporated herein by
reference in their entirety and for all purposes to the same extent
as if each individual publication or patent or patent application
was specifically and individually indicated to be incorporated by
reference in its entirety for all purposes.
[0255] Many modifications and variations of this invention can be
made without departing from its spirit and scope, as will be
apparent to those skilled in the art. The specific embodiments
described herein are offered by way of example only, and the
invention is to be limited only by the terms of the appended
claims, along with the full scope of equivalents to which such
claims are entitled.
TABLE-US-00001 TABLES 1 A B E F H I 1 left right C D dna
transposase G dna Confluence J K L 2 SEQ* SEQ* Int Seq system (ng)
SEQ* nls (ng) (0-100%) GFP1 GFP2 GFP3 3 35 36 5'-TTAA-3' piggyBac
500 none N/A 0 0 5 5 2 4 35 36 5'-TTAA-3' piggyBac 500 698 no 160
100 707 677 659 5 1 11 5'-TTAA-3' Xenopus 500 none N/A 0 0 4 3 3 6
1 11 5'-TTAA-3' Xenopus 500 49 yes 160 100 886 890 779 7 1 11
5'-TTAA-3' Xenopus 500 49 no 160 15 104 105 109 8 1 11 5'-TTAA-3'
Xenopus 500 48 yes 160 100 828 904 803 9 1 11 5'-TTAA-3' Xenopus
500 48 no 160 5 47 45 55 10 3 12 5'-TTAA-3' Xenopus 500 none N/A 0
0 5 5 6 11 3 12 5'-TTAA-3' Xenopus 500 49 yes 160 100 918 858 820
12 3 12 5'-TTAA-3' Xenopus 500 49 no 160 10 27 25 26 13 3 12
5'-TTAA-3' Xenopus 500 48 yes 160 100 953 933 921 14 3 12
5'-TTAA-3' Xenopus 500 48 no 160 10 73 76 65 15 23 29 5'-TTAT-3'
Bombyx 500 none N/A 0 0 2 4 4 16 23 29 5'-TTAT-3' Bombyx 500 750
yes 160 0 11 8 8 17 23 29 5'-TTAT-3' Bombyx 500 750 no 160 0 2 4 4
18 23 29 5'-TTAT-3' Bombyx 500 407 yes 160 100 1042 1089 1099 19 23
29 5'-TTAT-3' Bombyx 500 407 no 160 100 972 1046 960 *SEQ ID
NO.
TABLE-US-00002 TABLE 2 A B F G I left right C D E dna transposase H
dna J K L 1 SEQ* SEQ* Int Seq transposon system (ng) SEQ* nls (ng)
GFP1 GFP2 GFP3 2 23 29 5'-TTAT-3' 192465 Bombyx 500 no N/A 0 6 6 6
3 23 29 5'-TTAT-3' 192465 Bombyx 500 407 yes 160 817 788 705 4 24
29 5'-TTAT-3' 214228 Bombyx 500 no N/A 0 5 4 4 5 24 29 5'-TTAT-3'
214228 Bombyx 500 407 yes 160 600 591 602 6 25 29 5'-TTAT-3' 214229
Bombyx 500 no N/A 0 5 5 4 7 25 29 5'-TTAT-3' 214229 Bombyx 500 407
yes 160 103 119 127 8 23 31 5'-TTAT-3' 214230 Bombyx 500 no N/A 0 5
4 5 9 23 31 5'-TTAT-3' 214230 Bombyx 500 407 yes 160 13 12 11 10 22
30 5'-TTAA-3' 214404 Bombyx 500 no N/A 0 5 5 5 11 22 30 5'-TTAA-3'
214404 Bombyx 500 407 yes 160 1035 994 983 12 22 30 5'-TTAA-3'
214404 Bombyx 500 750 yes 160 4 4 3 13 3 12 5'-TTAA-3' 192462
Xenopus 500 no N/A 0 4 3 4 14 3 12 5'-TTAA-3' 192462 Xenopus 500 48
yes 160 1048 994 977 15 4 12 5'-TTAA-3' 214231 Xenopus 500 no N/A 0
4 5 4 16 4 12 5'-TTAA-3' 214231 Xenopus 500 48 yes 160 1346 1278
1269 17 5 12 5'-TTAA-3' 217099 Xenopus 500 no N/A 0 4 3 3 18 5 12
5'-TTAA-3' 217099 Xenopus 500 48 yes 160 964 872 901 19 3 13
5'-TTAA-3' 214233 Xenopus 500 no N/A 0 5 4 6 20 3 13 5'-TTAA-3'
214233 Xenopus 500 48 yes 160 1075 1014 1035 21 8 14 5'-TTAT-3'
214406 Xenopus 500 no N/A 0 4 3 4 22 8 14 5'-TTAT-3' 214406 Xenopus
500 48 yes 160 1205 1083 1058 23 35 36 5'-TTAA-3' 136214 piggyBac
500 no N/A 0 4 4 5 24 35 36 5'-TTAA-3' 136214 piggyBac 500 698 no
160 610 558 577 25 37 38 5'-TTAT-3' 214405 piggyBac 500 no N/A 0 4
3 3 26 37 38 5'-TTAT-3' 214405 piggyBac 500 698 no 160 485 464 451
27 N/A N/A N/A none negative 0 no N/A 0 4 5 5 *SEQ ID NO.
TABLE-US-00003 TABLE 3 A B F G I left right C D E dna transposase H
dna J K L 1 SEQ* SEQ* Int Seq reporter transposon (ng) SEQ* nls
(ng) GFP1 GFP2 GFP3 2 1 11 5'-TTAA-3' RC3 223949 500 no N/A 0 3 4 3
3 1 11 5'-TTAA-3' RC3 223949 500 48 yes 160 1157 1169 1095 4 6 11
5'-TTAA-3' RC3 223950 500 no N/A 0 3 3 4 5 6 11 5'-TTAA-3' RC3
223950 500 48 yes 160 788 930 887 6 7 11 5'-TTAA-3' RC3 223953 500
no N/A 0 3 4 3 7 7 11 5'-TTAA-3' RC3 223953 500 48 yes 160 965 957
918 8 1 15 5'-TTAA-3' RC3 223951 500 no N/A 0 4 4 4 9 1 15
5'-TTAA-3' RC3 223951 500 48 yes 160 1118 1136 1139 10 1 16
5'-TTAA-3' RC3 223952 500 no N/A 0 4 4 3 11 1 16 5'-TTAA-3' RC3
223952 500 48 yes 160 849 889 863 12 35 36 5'-TTAA-3' RC1 136214
500 no N/A 0 3 3 4 13 35 36 5'-TTAA-3' RC1 136214 500 698 no 160
536 555 572 14 N/A N/A N/A none none 0 no N/A 0 3 3 3 *SEQ ID
NO.
TABLE-US-00004 TABLE 4 Xenopus Transposase Bombyx Transposase A B C
D E F G H Position WT Transposition Excision Position WT
Transposition Excision 2 A RHL GKRPWIMNTEH 4 E T -n/a- 3 K VM
QLGWCA 9 R n/a P 4 R MCPK AIEM 12 A T PYQH 5 F CNQR CTHPVYEGN 13 M
P GQN 6 Y LHVICGASF RPDLNHVIC 14 L T -n/a- 7 S GV DG 15 E G -n/a- 8
A -n/a- ED 20 D G HWPQFGKV 9 E -n/a- WD 21 Y -n/a- K 10 E -n/a-
AIVNPDM 23 D -n/a- HPKS 11 A DV CD 24 E C YPT 12 A -n/a- YD 25 S
TKML YGKQL 13 A P TPK 26 S R FMWPYCTKA 14 H -n/a- PTG 27 S N
NCYAGHPLMT 15 C GI AGVDRLYI 28 E -n/a- AYCNIHPL 16 M ENDSQTA
ELHFINDS 30 E -n/a- HGW 17 A SYVLMT ESYVQL 32 D KQW INQGWFSYL 18 S
CYMLQGPAWHK ICYMVLQG 33 H S EGDK 19 S CVLFKEDGNAMPYRT CVLFQKEDG 36
E Q K 20 S GMLVHWACQDFN RGMLVHWACQD 37 H -n/a- WSDIQG 21 E
NWGQLDAPTSYV NWFGQLDM 39 V -n/a- A 22 E CHRLKSGMVQAYW CHRLKSDGMVQT
41 Y -n/a- SM 23 F QADWKTVMNPHECR QADYWKTVMNP 42 D -n/a- V 24 S
LWHVPIFKYDCNQ LWHGAVPIFKYDCN 43 T -n/a- YKIW 25 G N N 44 E CPAQ
ATNQHMDFIVL 26 S FHV FQHYWV 45 E SM KPCF 27 D LV L 46 E P APY 28 S
K YCMLHTQ 47 R -n/a- KM 29 E L LK 48 I A -n/a- 31 V -n/a- LTIQK 49
D K Y 32 P -n/a- SAV 50 S -n/a- A 33 P -n/a- VHS 55 S -n/a- A 34 A
-n/a- LE 58 R M -n/a- 35 S -n/a- ELM 62 A K RWCV 36 E -n/a- SVD 63
N T ETAW 37 S -n/a- DC 64 A P HTMV 38 D -n/a- FNA 65 I GQ -n/a- 39
S -n/a- VT 66 I E PWFT 40 S -n/a- T 67 A -n/a- DS 41 T -n/a- M 68 N
GHQ SPGVC 42 E KN KNPAS 69 E WQHTMPDLV QPHTDVC 43 E P QCWKGA 70 S
-n/a- D 44 S MEQ WLM 71 D KLMCNV KMNLY 45 W -n/a- LCVSPK 72 S FT
ETPDH 46 C EQTLHP MI 73 D -n/a- AHYECV 47 S NC FAN 74 P -n/a- VL 48
S VA VKWA 75 D -n/a- HAW 49 S G YLPVKTMDE 76 D AQGWVSC SAH 50 T DR
ANSK 77 D -n/a- TY 51 V QKYM TFH 78 L -n/a- I 52 S AT VAKPF 79 P KG
-n/a- 53 A VV EQYK 81 S -n/a- Q 54 L VPCE IANV 83 V -n/a- WQDF 55 E
HPK S 84 R NKHYW ENHYTW 56 E V LPYGQW 85 Q EMKHN TEKFLH 57 P VHTQ
KVSA 87 A MNKHYIC MNF 58 M IVPAKFL DIRVHN 88 S IY MV 59 E YAH MDN
89 A -n/a- KMYC 60 V -n/a- EQ 90 S -n/a- AD 62 E SCWVITLQFKG -n/a-
91 R -n/a- A 63 D TQP -n/a- 92 Q EAPNIYHFRDMWCGLV ADYNRGMFWHTVCPL
64 V IMQSKHFLTC -n/a- 93 V PKMFWL AIPQWFM 65 D MVPLKE -n/a- 94 S
EKTHCL KEIHYC 66 D GEAFW -n/a- 95 G EAQTKNMHDFLC ATD 67 L ATMVCHEY
-n/a- 96 P ATMRGVEQ EARCV 68 E SMYAPNVLQHD -n/a- 97 F QKHTCWVEPDARG
NADKTHRGYC 69 D RAPMLHVSW -n/a- 98 Y Q QF 70 Q CLTNSGH -n/a- 99 T N
DA 71 E PYMRWLF -n/a- 102 D -n/a- W 72 A EMTYQIGVFNKLCR -n/a- 103 G
-n/a- QM 73 G HNKFVDSWL -n/a- 107 Y -n/a- M 74 D T -n/a- 108 K
-n/a- S 75 R WCLMQ -n/a- 117 L -n/a- I 76 A LREIV -n/a- 122 I -n/a-
K 77 D QYLT -n/a- 128 Q -n/a- H 78 A QVGRC -n/a- 132 I -n/a- HDN 79
A FVR -n/a- 135 D -n/a- FM 80 A LY -n/a- 137 S -n/a- HIC 81 G STKV
-n/a- 139 E -n/a- P 82 G SLQWE -n/a- 140 Y -n/a- WQM 83 E FCHRDVN
-n/a- 145 I A RCAM 84 P SFGNVW -n/a- 149 S EHPQADT EQCAMKP 85 A MCR
-n/a- 150 D PEQ E 86 W G -n/a- 152 L GR -n/a- 87 G L -n/a- 153 Q
-n/a- HM 88 P AENHDL -n/a- 154 E Q DA 89 P HM -n/a- 157 T Q YCFE 90
C KDGNWVQTML -n/a- 160 N H -n/a- 91 N RALHV -n/a- 161 S N KN 92 F
YRGA -n/a- 162 S QWE WCE 93 P K -n/a- 164 R -n/a- Q 95 E QVNL -n/a-
165 H EGQTMVL CNDMKLQVWEA 96 I TW -n/a- 166 R CV CT 97 P HV -n/a-
167 Q -n/a- K 98 P R -n/a- 168 T YSWCNMGFALV IYMCWELKG 99 F SY
-n/a- 169 K HPSWGCMV HWYCEMLSV 100 T VL -n/a- 170 T HNG GQ 101 T
GFSVL -n/a- 171 A TG NYK 103 P RV -n/a- 172 A -n/a- CQ 104 G E
-n/a- 173 E QPA HQCLM 105 V F -n/a- 174 N -n/a- R 106 K RGME -n/a-
175 S NKG KG 107 V SR -n/a- 176 S HT HKT 108 D TH -n/a- 177 A -n/a-
IYCWMFVG 109 T IV -n/a- 178 E SHYFCAQGV YCDHLPWQSAVG 111 N F -n/a-
179 T Q HRK 114 P V -n/a- 180 S -n/a- YRKV 115 I Q -n/a- 182 Y
-n/a- H 116 N DQAF -n/a- 183 M K -n/a- 117 F L -n/a- 184 Q H YPG
118 F CSLQ -n/a- 185 E -n/a- K 119 Q HKS -n/a- 186 T I -n/a- 122 M
V VC 187 T D D 123 T L H 188 L I TG 124 E N SPQ 189 C DYIWTKMFPQ
IKQTV 125 A VNITPKLGS VQD 194 L AMVSTYC C 126 I CVLS -n/a- 195 I
FMV MV 127 L FMC V 196 A G G 128 Q K IE 198 L EQWTMI WT 129 D NI
EQL 200 L IFCM YICMF 130 M W -n/a- 201 A QLM MQ 132 L NFTHEMYQ
KNFTH 203 L VDGECTMA *YTCMA 133 Y T FH 204 I FACMTGV DCMNTG
136 V MITH FMDRN 205 K HR H 137 Y HAFNR HAFQSLN 206 S A -n/a- 138 A
G -n/a- 207 N GA G 139 E S AITVN 209 Q E YT 140 Q RN TR 210 S NC
-n/a- 141 Y IMQSEWVFACKLHR IMQSEWVF 211 L GMCTVA CTV 142 L
VFANQMIRKGYHW VFATNQCMIRK 212 K C -n/a- 143 T -n/a- AYV 213 D E E
144 Q RLMEGFDATV RNLMHPSECG 214 L IM IM 145 N CMAQIFGDEVHWY
CLRMAQSIFGDEV 215 W Y Y 146 P VTW CQLYKVNFE 216 R KA K 147 L
PQGKVTMFRI PWQHG 217 T VAIPCQM IFDQCAKV 148 P MRVFT MRVCFTQH 219 G
SAC CHAQ 149 R -n/a- LQGP 220 T -n/a- C 150 Y WAGFHSVCMNDEQK
WAGFHSV 222 V T A 151 A GS REGCS 223 D E NS 154 H -n/a- CL 224 I V
V 155 A QM -n/a- 227 T NI N 157 H YFT SWY 228 T -n/a- C 158 P VE
VGS 229 M F -n/a- 159 T -n/a- PR 234 F -n/a- Y 160 D -n/a- YWC 235
Q CNHGWYATEM AHCEWMTFG 161 I AVLQ AVLYHK 237 L I IV 162 A LVCKT
GMSLIYVCQ 238 Q CMHVL MTHIL 163 E -n/a- KGD 239 N GSA G 164 M -n/a-
E 240 N CHMA WSCAMH 165 K -n/a- RTFC 302 P K -n/a- 166 R -n/a- A
303 N CRG ADSHERKLQ 167 F -n/a- R 304 K -n/a- Y 168 V LTIM L 305 P
H -n/a- 169 G -n/a- D 306 A QC -n/a- 170 L -n/a- D 307 K R -n/a-
171 T A P 308 Y V -n/a- 172 L I SAR 310 I WML L 173 A LSG LMSIG 311
K L -n/a- 174 M ATQ WASG 312 I FCALTVGM CAMLV 175 G -n/a- APC 313 L
FQIEHCYMV IHQM 176 L -n/a- DM 314 A DQET T 177 I RVA RLV 315 L IVM
M 178 K -n/a- RG 316 V IA TC 179 A TKSVR TK 317 D C C 180 N -n/a-
TSQ 318 A TLECV CV 181 S -n/a- A 319 K CGNHMALQVSDT SDICATQV 182 L
VIQTWR SVI 320 N ALVRDTQCS RATGHCMVLK 184 S -n/a- Y 321 F
HRNYWDGEMKAQ NHKMYW 185 Y -n/a- T 322 Y F FM 187 D GI LMQNGFH 323 V
ILTM MIALT 188 T RQSMHIV RC 324 V NACILTKYHFSQM GIYHFAMTQLK 189 T
CNLKQVAWYGFS CNLKHQVAWYG 325 N -n/a- HCK 190 T C NW 326 L GCA AMC
191 V -n/a- AELMQI 327 E NQCHDWFLA NHMQT 192 L VCHM VICH 328 V TIMP
TAL 193 S PTRKGDNFH PTRKQGYDN 330 A KVP SPCTLV 194 I VP LHRGCV 331
G -n/a- A 195 P G SGR 332 K -n/a- CQ 196 V LSWAF MI 333 Q PTMH SM
197 F -n/a- SML 334 P H -n/a- 198 S R AK 335 S HTYKMAGCQLV
NPKYMAECHTQV 199 A HGNCKRQWSM HGNCIKR 336 G PVS -n/a- 200 T
CIMLNWVQYH CRIMLNWV 337 P WEHIAMNDKQ DGSCKMALV 201 M -n/a- C 339 A
G -n/a- 202 S A PA 340 V G -n/a- 203 R -n/a- V 341 S NCPA -n/a- 204
N -n/a- PT 342 N Q -n/a- 205 R -n/a- L 343 R SKG -n/a- 206 Y -n/a-
P 344 P GNCSA GSNA 207 Q -n/a- T 345 F STAQGC PAKMC 208 L Q PG 346
E IQN -n/a- 209 L IM IMA 347 V L -n/a- 210 L H A 349 E TD TG 211 R
TCQASK TCQASK 352 I V -n/a- 212 F YNM CAYN 353 Q NET -n/a- 213 L
-n/a- PM 355 V F -n/a- 214 H -n/a- NYMQASE 356 A R -n/a- 215 F WE Q
357 R W -n/a- 216 N -n/a- Q 359 H -n/a- GC 217 N E Q 361 N TCQM VCM
218 N VRTC VRGIPDE 362 V -n/a- L 219 A WGEV DTLQWIMY 365 D YKT T
220 T EI ADEL 367 W YF -n/a- 221 A M VC 368 F -n/a- Y 222 V TILK
QPTILSK 369 T SA -n/a- 223 P -n/a- TS 370 G HQ -n/a- 224 P QDS
QMDVR*EK 371 Y -n/a- P 225 D KYL KEPGRMAN 372 E -n/a- PT 226 Q R AP
373 L VIST TI 227 P VDASTNF VHGDAES 374 M G -n/a- 228 G -n/a- HTRQ
375 L C -n/a- 229 H LP VD 376 H YAK Q 230 D -n/a- Q 379 N GA -n/a-
231 R P -n/a- 380 E WC T 233 H PV FPW 381 Y -n/a- HL 234 K -n/a-
ALCDVE 382 R NK K 235 L IV IV 385 S -n/a- -n/a- 236 R -n/a- Q 386 V
TICL TLIC 237 P Q -n/a- 387 G -n/a- S 238 L -n/a- VDN 388 T -n/a- V
239 I V L 389 V IMTL MAL 240 D N HVR 391 K -n/a- IMTPL 242 L
IAE*FRS NWIAE 392 N RFV HFVQ 243 S GTLQ GT 394 R HKT PMTA 244 E
RNHLMQ RFVDNHLM 395 Q PFECVA HSYPA 245 R QITECP QKNI 398 E -n/a- QA
246 F SRL SR 399 S NEKHDYGQRTAV KMQG 247 A RECSQHV RECGSNQ 400 F
GCPWLYM YWM 248 A SLHCNIQY SLMHCDN 401 I VCKT WK 249 V TPIAY TPMID
402 R YKDFGNEMSQTCLV SEQFK 250 Y PHT PHT 403 T WAVFLYNGCISMQK
NFGICEQVL 251 T ISKVLMQD ISNYKVR 404 D ISENHCMGAQLPV WMESFANLGVQP
252 P L L 405 R NTL G 253 C RTLHNGDQVM RTLHNGDQ 406 Q FG ICE 254 Q
MVL RH 407 P KTIQMV K 255 N -n/a- A 408 N FIAEMSDYHCQVWL IAPEKLHV
256 I VC V 409 S HYNIDFTC QDNT 257 C VYR V 410 S THY TC 258 I DRH
DRH 411 V EQHDS -n/a- 259 D TR TR 412 F AW -n/a- 260 E V V 414 F W
-n/a- 261 S A A 415 Q N AN 262 L SA SA 416 K -n/a- S 263 L VAMRD
VAM 418 I C -n/a- 264 L VPDKMR VS 419 T FICS -n/a- 265 F YK EHYW
420 L M -n/a- 266 K GRA GR 424 A -n/a- D 267 G PL P 426 K -n/a- T
268 R A ACHYQK 428 N S H
269 L SIVCQ SIVC 430 V -n/a- D 270 Q VKACPLIEGYNTW VHKACPL 432 V Y
THMC 271 F VPT VP 433 V -n/a- L 272 R KILSVC K 434 M Q A 273 Q MVE
MVITN 436 T -n/a- S 274 Y HI H 440 D SCMLV SIKCAQ 275 I PLM PL 441
N FRMGCDL GFAVLW 276 P IRAWCL IR 442 S YKFVL KGFCYWV 277 S EAK EA
443 I EFV AYK 278 K AL A 444 D QIMV MA 279 R YQKVGS Y 445 E
PYHCGKMQL CGMKPNLTW 280 A S S 446 S -n/a- EAMDYCPLWG 281 R LY LYK
447 T -n/a- QS 282 Y LQGCVHSNT LQGCEVH 448 G WYHCTV NWQ 283 G YI
YAI 449 E APTL HGTCIL 284 I QVGLF QV 450 K -n/a- T 285 K I I 451 Q
VENDSRYHGFCITPMWL ENTRCSMYAW 286 F LT L 452 K IFVL -n/a- 287 Y
QKSFW QKSF 454 E -n/a- C 288 K TADFLC T 455 M QCLV PGCVI 289 L
CTRGYVE CTR 456 I ACMLTV V 290 C TVQ T 457 T CGA A 291 E VD VCND
458 F ADC A 292 S RVA R 461 S KGEDYA KTL 293 S NDHTWAK NDHTWAK 464
A S T 294 S RNGT RCNGT 466 V TC C 295 G TDSL TDS 468 V QMT CT 296 Y
HF HF 469 V TAHCL HACT 297 T CPVMLD CPVML 471 E Q -n/a- 298 S
EVMKGLNCQA EVMKGLNCQA 472 L KQM -n/a- 299 Y HKCREGAN HKCREGA 473 C
GSQT IGSTM 300 F YM VCIYM 474 A CQMGT CTV 301 L -n/a- -n/a- 475 N
-n/a- S 302 I -n/a- V 477 N -n/a- D 304 E H DHSQC 483 K R N 305 G
-n/a- E 484 R -n/a- HK 306 K -n/a- NL 485 W FYTDKEQMV -n/a- 307 D
-n/a- F 486 P -n/a- EMA 308 S -n/a- RG 488 T KV V 309 K -n/a-
GCHMLQE 489 L YIV CTV 310 L R IRV 491 Y V -n/a- 311 D -n/a-
FHYWSNRILC 492 G A A 312 P -n/a- C 493 V HQWMIL IML 313 P SMLY
VSFKMHE 496 M D L 314 G ASIHL NQM 499 I DHWTCEMALV CWV 315 C -n/a-
SR 502 C SYML -n/a- 316 P N RDA 503 I MALQF QFL 317 P DLK
DNFMHCGVALKE 505 Y -n/a- Q 318 D RL NTAFKQHRCWEM 507 T RDSGKIMECAL
DIMECAVL 319 L CVF CIDVAM 509 K H -n/a- 320 T CGSNKHMV CGSRNKQ 510
N KGA QGK 321 V I INT 511 V KA CTEKA 322 S -n/a- ICT 512 T MA MCA
323 G -n/a- A 513 I V M 324 K G SR 514 K -n/a- P 325 I -n/a- L 515
R K -n/a- 326 V -n/a- WT 516 T -n/a- S 327 W -n/a- M 517 E DA NKAQ
328 E SHKVWFQLT SYIHRKVWADFMCQ 521 S HKQGE HCGETK 329 L -n/a- GM
523 G QTAMSC TMSICLA 330 I AV M 524 L KM HIYM 331 S AGQP AKWNDRG
525 S Q CNDTQ 332 P Q KGD 527 I MV M 333 L M WFM 528 Y NWMQKV
IGDNAQMER 334 L V VMC 529 E D -n/a- 335 G K LCNEA 531 L -n/a- M 336
Q YNMATL YNIGFEMVCH 532 H M CV 338 F SHP SYH 533 S IMELVA WMIQEVA
339 H -n/a- QR 535 N GVL SCMFVL 340 L V V 536 K A -n/a- 342 V -n/a-
GC 537 K M -n/a- 343 D AN -n/a- 539 N HGC -n/a- 344 N MGS RQTMG 540
I FM M 345 F G YW 542 T CAR HRK 346 Y S S 543 Y CWI MQACRH 347 S V
T 545 R -n/a- H 348 S -n/a- T 546 Q F C 349 I -n/a- V 549 E KCIQA
HCMQSFLA 351 L H R 550 K RMQ -n/a- 352 F C -n/a- 551 Q A -n/a- 353
T -n/a- SCV 552 L -n/a- I 354 A VWD VCRWEKHG 553 G TA HP 355 L T
-n/a- 554 E D YCL 356 Y C H 555 P ED YDC 357 C QHWNIVMRF DQHWNIV
556 S GV I 358 L AFERQVHCMY AFKERQNIVH 557 P WTSAQK DKGNLV 359 D
ALHRSQE ALHMR 558 R K SMQ 360 T P PLYSP 559 H KSC SIWK 361 P -n/a-
QS 560 V FPI HYKIP 362 A -n/a- P 561 N P QGA 363 C -n/a- W 562 V Y
ISM 364 G -n/a- D 563 P ITKE DE 366 I -n/a- L 564 G L QPCF 367 N
-n/a- TGR 565 R K -n/a- 368 R -n/a- TAWKP 566 Y M -n/a- 369 D -n/a-
RVAQSLML 567 V IH N 371 K -n/a- A 570 Q F N 372 G -n/a- FY 571 D
SFVQM NSMTAV 373 L -n/a- MI 573 P K MT 375 R -n/a- SQKAVPT 574 Y V
A 376 A -n/a- TCLVEIMGK 575 K -n/a- H 377 L -n/a- VI 576 K W I 378
L -n/a- ITMCKYV 581 K H -n/a- 379 D -n/a- A 583 S M -n/a- 380 K
-n/a- LAFTVE 585 N -n/a- SKGL 381 K -n/a- LPVRITN 586 A E NH 382 L
-n/a- VC 588 A GRF -n/a- 383 N -n/a- LIDFPVESGAHK 593 M -n/a- I 384
R -n/a- C 594 E C -n/a- 385 G -n/a- NWAMCHYSKQ 597 K -n/a- W 387 T
-n/a- RE 598 F M -n/a- 388 Y -n/a- FV 599 L Y -n/a- 389 A -n/a- YF
601 E V FQW 390 L -n/a- V 602 N GRQHTEDS GTQMEH 392 K -n/a- YMWC
603 C D -n/a- 393 N -n/a- EA 604 A I DTSI 394 E -n/a- F 605 E
RWKMPYCHAQSV PYAMRWHQVIGK 397 A -n/a- SIFLCVM 606 L VQYAEGCKNHM
EQCYANWMVK 399 K -n/a- ASH 607 D VYCNWTAHQELKG HQTYWCANLEKG 400 F
-n/a- C 608 S EDRM QWRCV 401 F -n/a- YD 609 S RWHVQGTKN WHTGNYKV
402 D -n/a- G 610 L TIKGAWDQSFN DSI 405 N -n/a- VD 406 L -n/a- GTM
409 L -n/a- I
422 R G QLKWSM 423 V NPTFHCS GARLNPTFHC 424 G CNSL CKQYPWNTHS 425 E
-n/a- SAQCAPGH 426 P LKYF TWLVCSQHKYN 428 K RQ NTFR 429 N GPYM
GWPYEHRAMS 430 K R QPR 431 P LQ LTC 432 L TMF HTSQMN 434 S A AT 435
K LTR YMHISVLA 436 E QAMLY WHCQAFML 438 S QA QMA 439 K LMR HALMCR
440 Y FLT WQFLH 442 G -n/a- W 443 G V V 444 V -n/a- C 446 R LMK
HTLMK 447 T SAC QSNAGC 450 L MVA MIVE 451 Q LMF ALMF 452 H -n/a- AS
455 N D E 457 T -n/a- C 458 R -n/a- * 460 T S A 461 R YQKT Y 462 A
MTYFKRQHE MTYNFCKRQH 464 Y -n/a- QW 465 K VHM VHT 467 V TCA KTC 468
G SF CST 469 I V NV 470 Y H -n/a- 471 L VM FCVM 472 I VLW VMLFW 473
Q -n/a- D 474 M AT IQAT 475 A STG -n/a- 476 L M IVNFMCQ 477 R AQ HV
478 N -n/a- K 479 S L L 480 Y HF H 482 V L L 483 Y T T 484 K G
GEAFVS 485 A CQV C 486 A EHCV R 487 V NTR NCMW 488 P EHKQFM ELND
489 G -n/a- YFQ 490 P GTHAKL GTCIMH 491 K QVGCLM QVIGW 492 L -n/a-
VQ 493 S ATPI GA 494 Y F M 495 Y FL MF 496 K H VQ 497 Y T -n/a- 498
Q CM VLGHTCEM 499 L HACVQTRNW HGACVQKTR 500 Q ECRHA ETCRFMVH 501 I
LMVS TLM 502 L IMVG FIMV 503 P HENCASLQ HENCV 504 A
NMVIPWDQLTKGFHYS NMVIPWDQLTK 505 L MC M 506 L MIC HQMI 507 F VWHMK
IV 508 G QTYR IS 509 G NLRMKQHIPCFA TNWLRMKQ 510 V MCAN HKM 511 E
TMILP TYQMF 512 E SYMKVARLTI SGP 513 Q YFVNISKW YFVPMAE 514 T
QVHFMRP QWNVHFMGR 515 V FTRAL KFHS 516 P -n/a- LM 517 E MVAKL
MGVASI 518 M SHLFTA IRSHLWFVG 519 P WR FWMND 520 P WRMFQVGDKY
WERMLTFQVGDK 521 S AHCVW TKFAHG 522 D AR VNEAFH 523 N WAGSPM
WQALKGDHSFC 524 V PMA PH 525 A QLIR QMLNI 527 L VHMRANFW
SYVHTCMRAIQ 528 I LKVFQHNTG RLKVFAMQHY 529 G -n/a- MAHDLVWC 530 K
QG MVQR 531 H RP -n/a- 532 F CMVQ CIHYRNMKVTA 533 I VF MVTSFGE 534
D EQLRVCMNAGF THEQLKRVCMNSA 535 T SRALV SRCAFLGVHKINMW 536 L Q
DMIQHSRKEVF 537 P -n/a- NF 538 P -n/a- TAFGVYKW 539 T SNL SMKQI 540
P KH KEVRMFN 541 G -n/a- K 542 K -n/a- QNYHT 543 Q -n/a- T 544 R
-n/a- FD 545 P -n/a- T 546 Q T TAVYN 547 K -n/a- T 548 G -n/a- DE
549 C -n/a- AY 550 K N NPCSFLT 551 V -n/a- AYM 552 C -n/a- H 553 R
K NMTVHK 554 K VT VMACLGYFIPE 555 R HV ELKHVGDIFMTN 556 G SCN
VMFDSKCQA 557 I KFSV RNHQLKPFCG 558 R G GL 559 R H HIVLTGKEYSWMFQN
560 D G TRLHSVMAGNC 561 T V SVAQI 562 R -n/a- VK
563 Y -n/a- AFNSGR 564 Y TV GMFTNQ 565 C -n/a- V 566 P VHKQ VHGASMK
567 K ML MLQVT 568 C -n/a- W 569 P YT VLYSEMTF 570 R F VLMTYK 571 N
VDMK FVWTDMYK 572 P -n/a- VNQF 573 G -n/a- RCA 574 L M TMIP 575 C H
H 576 F LKVDWMCR LKQAVYDWNMGCIER 577 K LGDRHYI EVLGDRNHYI 578 P
SENTQVM SCKENGID 579 C -n/a- Y 580 F M EAH 581 E IWRSGVHAC
ILWRFQSGDTVM 582 I VKRMG NEVKAQ 583 Y L CFDQ 584 H -n/a- L 585 T G
QHANCY 586 Q LCRYHFENKGAW LVTDMCRYHFENKGA 587 L FDRIPNESYMQGWKT
FDRIPNESYMQGWKT 588 H RK SMWRGE 589 Y VCKMIEDQR SVCFHKNWGPMI
TABLE-US-00005 TABLE 5 A B D E G H I left right C DNA transposase F
transposase DNA RNA J K L 1 SEQ* SEQ* transposon (ng) SEQ* nls
promoter (ng) (ng) GFP1 GFP2 GFP3 2 35 36 136214 500 no N/A N/A 0 0
4 5 6 3 35 36 136214 500 698 no N/A 160 0 468 458 476 4 1 11 202970
500 no N/A N/A 0 0 6 6 5 5 1 11 202970 500 48 yes CMV 160 0 1079
1086 1137 6 1 11 202970 500 48 yes PGK 160 0 248 269 244 7 1 11
202970 500 48 yes UBB 160 0 179 188 196 8 1 11 202970 500 48 yes
SV40 160 0 1305 1247 1293 9 1 11 202970 500 48 yes N/A 0 125 74 78
78 10 1 11 202970 500 48 yes N/A 0 250 262 252 249 11 1 11 202970
500 48 yes N/A 0 500 328 347 342 12 1 11 202970 500 48 yes N/A 0
1000 45 40 45 13 3 12 192462 500 no N/A N/A 0 0 5 6 6 14 3 12
192462 500 48 yes CMV 160 0 820 873 915 15 3 12 192462 500 48 yes
PGK 160 0 44 42 40 16 3 12 192462 500 48 yes UBB 160 0 29 31 31 17
3 12 192462 500 48 yes SV40 160 0 1535 1523 1537 18 3 12 192462 500
48 yes N/A 0 125 13 14 15 19 3 12 192462 500 48 yes N/A 0 250 97
113 115 20 3 12 192462 500 48 yes N/A 0 500 283 271 277 21 3 12
192462 500 48 yes N/A 0 1000 31 31 30 N/A N/A N/A 0 no N/A N/A 0 0
2 5 4 *SEQ ID NO.
TABLE-US-00006 TABLE 6 A B C E F H I left reporter right D DNA
transposase G DNA RNA J K L 1 SEQ* SEQ* SEQ* transposon (ng) SEQ*
nls (ng) (ng) GFP1 GFP2 GFP3 2 35 39 36 136214 500 no N/A 0 0 7 7 9
3 35 39 36 136214 500 698 no 160 0 57 59 60 4 23 39 29 192465 500
407 yes 0 0 6 6 5 5 23 39 29 192465 500 407 yes 0 125 37 35 35 6 23
39 29 192465 500 407 yes 0 250 786 783 792 7 23 39 29 192465 500
407 yes 0 500 903 908 934 8 23 39 29 192465 500 407 yes 0 1000 184
198 225 9 23 39 29 192465 500 407 yes 125 0 311 322 336 10 23 39 29
192465 500 407 yes 250 0 254 260 272 11 23 39 29 192465 500 407 yes
500 0 174 176 193 12 23 40 29 194093 500 no N/A 0 0 884 911 936 13
23 40 29 194093 500 407 yes 0 125 2861 2533 2830 14 23 40 29 194093
500 407 yes 0 250 4123 3907 4074 15 23 40 29 194093 500 407 yes 0
500 5668 5564 5554 16 23 40 29 194093 500 407 yes 0 1000 7387 7062
7355 17 23 40 29 194093 500 407 yes 125 0 7863 7281 7000 18 23 40
29 194093 500 407 yes 250 0 7684 8043 8335 19 23 40 29 194093 500
407 yes 500 0 8201 7826 7684 20 N/A N/A N/A N/A 0 no N/A 0 0 4 4 4
*SEQ ID NO.
TABLE-US-00007 TABLE 7 A E F H I J Linker B C D Promoter Intron G
intergenic Enhancer Promoter Row SEQ* Gene GFP RFP 1 1 polyA1
insulator 2 2 1 N/A 188550 no yes EF1a EF1a globin (rabbit) N/A N/A
N/A 2 N/A 181650 yes no EF1a EF1a globin (rabbit) N/A N/A N/A 3 N/A
146674 yes yes EF1a EF1a N/A N/A N/A N/A 4 1051 188209 yes yes EF1a
EF1a N/A N/A N/A N/A 5 1062 206694 yes yes EF1a EF1a N/A N/A N/A
N/A 6 1063 206695 yes yes EF1a EF1a N/A N/A N/A N/A 7 1064 206696
yes yes EF1a EF1a N/A N/A N/A N/A 8 1011 203906 yes yes EF1a EF1a
HSV-TK/gastrin no CMV EF1a 9 1023 203907 yes yes EF1a EF1a
HSV-TK/gastrin no CMV actin 10 1025 203909 yes yes EF1a EF1a
HSV-TK/gastrin no no EF1a 11 998 203910 yes yes EF1a EF1a
HSV-TK/gastrin no CMV CMV 12 1002 203914 yes yes EF1a EF1a
HSV-TK/gastrin 2xHS4c CMV GAPDH 13 1000 203912 yes yes EF1a EF1a
HSV-TK/gastrin no CMV GAPDH 14 1001 203913 yes yes EF1a EF1a
HSV-TK/gastrin 2xHS4c no EF1a 15 1024 203908 yes yes EF1a EF1a
HSV-TK/gastrin no no EF1a 16 1003 203915 yes yes EF1a EF1a
HSV-TK/gastrin 2xHS4c CMV CMV 17 999 203911 yes yes EF1a EF1a
HSV-TK/gastrin no CMV CMV 18 1004 203916 yes yes EF1a EF1a
HSV-TK/gastrin 2xHS4c no EF1a 19 1005 203917 yes yes EF1a EF1a
HSV-TK/gastrin no no EF1a 20 1006 203918 yes yes EF1a EF1a
HSV-TK/gastrin 2xHS4c CMV GAPDH 21 1007 203919 yes yes EF1a EF1a
HSV-TK/gastrin no CMV GAPDH 22 1008 203920 yes yes EF1a EF1a
HSV-TK/gastrin 2xHS4c CMV GAPDH 23 1009 207390 yes yes EF1a EF1a
HSV-TK/gastrin no CMV GAPDH K Intron L M N O P Q Row 2 pA2 GFP RFP
R/G ORF2/ORF1 D % 1 N/A N/A 4 1564 N/A N/A 0.00 2 N/A N/A 8542 2
N/A N/A 1.00 3 N/A globin (rabbit) 1508 322 0.21 1.00 0.18 4 N/A
globin (rabbit) 9964 741 0.07 0.33 1.17 5 N/A globin (rabbit) 6248
604 0.10 0.48 0.73 6 N/A globin (rabbit) 6206 529 0.09 0.43 0.73 7
N/A globin (rabbit) 6280 586 0.09 0.43 0.74 8 no globin (rabbit)
1830 117 0.06 0.29 0.21 9 no globin (rabbit) 1982 97 0.05 0.24 0.23
10 no globin (rabbit) 2714 80 0.03 0.14 0.32 11 CMVc globin
(rabbit) 1613 565 0.35 1.67 0.19 12 GAPDH globin (rabbit) 2432 688
0.28 1.33 0.29 13 eMLP globin (rabbit) 2150 316 0.15 0.71 0.25 14
EF1a globin (rabbit) 2853 1504 0.53 2.52 0.33 15 EF1a globin
(rabbit) 2795 252 0.09 0.43 0.33 16 no globin (rabbit) 2505 142
0.06 0.29 0.29 17 no globin (rabbit) 1012 118 0.12 0.57 0.12 18
eMLP globin (rabbit) 3430 537 0.16 0.76 0.40 19 eMLP globin
(rabbit) 2390 185 0.08 0.38 0.28 20 CMVc globin (rabbit) 1903 533
0.28 1.33 0.22 21 CMVc globin (rabbit) 2169 310 0.14 0.67 0.25 22
no globin (rabbit) 2046 410 0.20 0.95 0.24 23 no globin (rabbit)
2087 226 0.11 0.52 0.24 *SEQ ID NO.
TABLE-US-00008 TABLE 8 B C D E F G H I J K L A Transposon Linker
Trans- Trans- Enhancer Promoter Intron intergenic Enhancer Promoter
Row SEQ* poson 1 poson 2 GFP RFP 1 1 1 polyA1 insulator 2 2 1 N/A
187151 N/A yes no CMV CMV none globin (rabbit) N/A N/A N/A 2 N/A
187151 N/A yes no CMV CMV none globin (rabbit) N/A N/A N/A 3 1051
188209 N/A yes yes none EF1a EF1a N/A N/A none none 4 1051 188209
N/A yes yes none EF1a EF1a N/A N/A none none 5 1054 188219 N/A yes
yes none EF1a EF1a N/A N/A none none 6 1054 188219 N/A yes yes none
EF1a EF1a N/A N/A none none 7 998 198833 N/A yes yes CMV CMV none
HSV-TK none CMV CMV 8 998 198833 N/A yes yes CMV CMV none HSV-TK
none CMV CMV 9 999 198834 N/A yes yes CMV CMV none HSV-TK none CMV
CMV 10 999 198834 N/A yes yes CMV CMV none HSV-TK none CMV CMV 11
1000 198835 N/A yes yes CMV CMV none HSV-TK none CMV GAPDH 12 1000
198835 N/A yes yes CMV CMV none HSV-TK none CMV GAPDH 13 1001
198836 N/A yes yes CMV CMV none HSV-TK 2x HS4 none EF1a core 14
1001 198836 N/A yes yes CMV CMV none HSV-TK 2x HS4 none EF1a core
15 1002 198837 N/A yes yes CMV CMV none HSV-TK 2x HS4 CMV GAPDH
core 16 1002 198837 N/A yes yes CMV CMV none HSV-TK 2x HS4 CMV
GAPDH core 17 1003 198838 N/A yes yes CMV CMV none HSV-TK 2x HS4
CMV CMV core 18 1003 198838 N/A yes yes CMV CMV none HSV-TK 2x HS4
CMV CMV core 19 N/A 200967 N/A no yes CMV CMV none globin (rabbit)
N/A N/A N/A 20 N/A 200967 N/A no yes CMV CMV none globin (rabbit)
N/A N/A N/A 21 N/A 187151 200967 yes yes CMV CMV none globin
(rabbit) N/A N/A N/A 22 N/A 187151 200967 yes yes CMV CMV none
globin (rabbit) N/A N/A N/A M N Transposon P Q R S T U Intron O GFP
Expression RFP Expression Row 2 pA2 Transposase 1 2 3 1 2 3 1 N/A
N/A no 70 66 65 2 2 2 2 N/A N/A yes 1250 1083 1330 1 2 1 3 none
globin (rabbit) no 706 660 698 62 60 66 4 none globin (rabbit) yes
6764 4922 5238 643 467 480 5 none globin (rabbit) no 307 370 375 32
38 36 6 none globin (rabbit) yes 3656 4019 4243 407 452 474 7 CMVc
globin (rabbit) no 20 17 17 15 12 17 8 CMVc globin (rabbit) yes 87
94 99 113 120 126 9 none globin (rabbit) no 19 22 21 9 10 10 10
none globin (rabbit) yes 152 128 141 64 56 62 11 eMLP globin
(rabbit) no 26 32 27 17 17 18 12 eMLP globin (rabbit) yes 272 231
222 306 257 237 13 EF1a globin (rabbit) no 38 39 36 104 94 98 14
EF1a globin (rabbit) yes 320 374 449 1102 1245 1471 15 GAPDH globin
(rabbit) no 67 55 55 58 45 42 16 GAPDH globin (rabbit) yes 396 470
411 418 483 425 17 none globin (rabbit) no 25 27 22 11 13 10 18
none globin (rabbit) yes 280 260 245 122 118 104 19 N/A N/A no 5 5
4 4 10 11 20 N/A N/A yes 5 6 6 375 389 392 21 N/A N/A no 34 33 35 7
8 8 22 N/A N/A yes 546 583 628 186 196 197 *SEQ ID NO.
TABLE-US-00009 TABLE 9 B C D E F G H I A HEK HEK HEK HEK # CHO CHO
CHO CHO # IRES RFP/ IRES GFP measure- RFP/ IRES GFP measure- Row
SEQ* GFP efficiency % ments GFP efficiency % ments 1 1050 0.07 0.22
0.39 3 0.08 0.28 0.15 4 2 1051 0.12 0.41 0.44 4 0.11 0.38 0.29 4 3
1052 0.10 0.35 0.25 4 0.04 0.13 0.47 5 4 1053 0.05 0.17 0.39 3 0.05
0.17 0.25 4 5 1065 0.08 0.27 0.26 2 0.09 0.30 0.14 2 6 1066 0.07
0.23 0.51 4 0.02 0.08 0.62 4 7 1067 0.00 0.01 0.31 2 0.01 0.05 0.21
2 8 1068 0.08 0.25 0.48 3 0.02 0.06 0.58 4 9 1069 0.03 0.08 0.21 1
0.01 0.02 0.39 1 10 1070 0.01 0.04 0.15 1 0.00 0.01 0.26 1 11 1071
0.13 0.45 0.46 3 0.09 0.28 0.54 3 12 1072 0.07 0.23 0.16 2 0.02
0.08 0.58 3 13 1073 0.06 0.19 0.58 2 0.07 0.24 0.25 3 14 1074 0.03
0.10 0.54 1 0.04 0.14 0.38 2 15 1075 0.12 0.39 0.37 2 0.05 0.15
0.72 3 16 1076 0.11 0.35 0.32 1 0.04 0.15 0.79 2 17 1077 0.03 0.11
0.33 1 0.01 0.05 0.66 2 18 1078 0.03 0.10 0.32 1 0.02 0.06 0.85 1
19 1079 0.07 0.22 0.61 2 0.03 0.12 0.64 1 20 1080 0.07 0.22 0.51 2
0.04 0.12 0.84 1 21 1081 0.11 0.35 0.32 1 0.04 0.12 0.67 1 22 1082
0.06 0.22 0.56 2 0.08 0.25 0.35 2 23 1083 0.08 0.27 0.40 2 0.10
0.33 0.25 2 24 1084 0.05 0.16 0.66 1 0.06 0.21 0.47 1 25 1085 0.04
0.13 0.57 1 0.04 0.14 0.30 1 26 1086 0.11 0.35 0.30 2 0.05 0.15
0.82 3 27 1087 0.08 0.27 0.42 2 0.09 0.28 0.16 2 28 1088 0.11 0.36
0.41 2 0.04 0.15 0.66 3 29 1089 0.02 0.06 0.38 1 0.02 0.05 0.85 1
30 1090 0.02 0.07 0.33 1 0.01 0.03 0.41 1 31 1091 0.00 0.01 0.21 1
0.00 0.01 0.63 1 32 1092 0.02 0.07 0.26 1 0.03 0.11 0.35 1 33 1093
0.07 0.23 0.25 3 0.03 0.10 0.58 4 34 1094 0.00 0.01 0.17 2 0.01
0.04 0.60 3 35 1096 0.06 0.18 0.29 1 nd nd nd 0 *SEQ ID NO.
TABLE-US-00010 TABLE 10 B C plasmid Transposase D E A config-
promoter DNA outgrowth F 1 Construct uration SEQ* (ug) 5 hours 24
hours 2 N/A N/A N/A 0 0 0 3 251587 circular 949 0.2 0 48 4 251587
circular 949 1 11 93 5 251587 circular 949 2 26 276 6 251588
circular 954 0.2 13 58 7 251588 circular 954 1 60 221 8 251588
circular 954 2 137 456 9 251589 circular none 0.2 2 0 10 251589
circular none 1 0 1 11 251589 circular none 2 1 6 12 251589 linear
none 1 661 ~1000 *SEQ ID NO.
TABLE-US-00011 TABLE 11 A E F J Xenopus B C D Weight Bombyx G H I
Weight Position From To Weight Std Position From To Weight Std 6 Y
C 0.09 0.03 85 Q E -0.01 0.03 7 S G 0.25 0.05 92 Q A 0.09 0.03 9 E
D 0.00 0.01 92 Q L -0.06 0.08 16 M S 0.23 0.05 92 Q N -0.04 0.02 18
S G -0.03 0.05 93 V L 0.35 0.08 19 S G 0.05 0.02 93 V M 0.20 0.09
20 S D 0.20 0.02 96 P G 0.07 0.02 20 S G 0.26 0.03 97 F C 0.03 0.03
20 S Q 0.40 0.05 97 F H 0.18 0.03 21 E D 0.38 0.07 165 H E 0.28
0.07 22 E Q 0.17 0.05 165 H W 0.27 0.07 23 F P 0.25 0.07 178 E H
0.13 0.06 23 F T 0.37 0.10 178 E S 0.29 0.04 24 S Y 0.17 0.05 189 C
P 0.12 0.08 26 S V 0.10 0.05 196 A G 0.48 0.02 28 S Q 0.10 0.03 200
L F -0.10 0.08 31 V K 0.04 0.02 200 L I 0.46 0.05 34 A E 0.03 0.02
200 L M 0.01 0.02 67 L A 0.10 0.04 201 A Q 0.22 0.10 73 G H 0.29
0.06 203 L T -0.03 0.11 76 A V 0.15 0.04 207 N G -0.01 0.07 77 D N
0.11 0.02 211 L A 0.20 0.03 88 P A 0.05 0.02 215 W Y 0.19 0.03 91 N
D 0.14 0.06 217 T A -0.05 0.02 141 Y A 0.14 0.03 217 T K 0.00 0.08
141 Y Q 0.33 0.04 219 G A -0.04 0.04 145 N E 0.03 0.02 219 G S 0.02
0.03 145 N V 0.02 0.03 235 Q G 0.13 0.08 146 P K 0.10 0.03 235 Q N
-0.06 0.09 146 P T 0.11 0.04 235 Q Y 0.33 0.08 146 P V 0.11 0.03
238 Q L 0.51 0.08 148 P H 0.03 0.02 242 R Q -0.06 0.06 148 P T 0.42
0.04 246 K I 0.24 0.05 150 Y C 0.10 0.05 253 K V 0.32 0.10 150 Y G
0.25 0.05 258 M V 0.18 0.06 150 Y S 0.21 0.04 261 F L 0.15 0.05 157
H Y 0.37 0.06 263 S K 0.28 0.07 162 A C 0.18 0.06 271 C S 0.36 0.04
179 A K 0.36 0.04 303 N R 0.11 0.07 182 L I 0.27 0.06 312 I V -0.02
0.08 182 L V 0.16 0.08 321 F D 0.12 0.06 189 T G 0.04 0.03 321 F W
0.18 0.08 192 L H 0.01 0.02 323 V T 0.01 0.02 193 S K 0.03 0.05 324
V H 0.28 0.07 193 S N 0.03 0.03 324 V K 0.32 0.08 196 V I 0.03 0.02
330 A V 0.34 0.09 198 S G 0.26 0.04 333 Q M 0.00 0.04 200 T W 0.02
0.02 337 P A -0.02 0.03 202 S A -0.01 0.06 368 F Y -0.08 0.10 210 L
H 0.15 0.05 373 L C 0.25 0.06 212 F N 0.17 0.09 373 L V 0.10 0.04
218 N E 0.11 0.06 389 V L 0.15 0.05 248 A N 0.50 0.05 394 R T -0.01
0.11 263 L M 0.35 0.06 395 Q P -0.11 0.10 270 Q L 0.07 0.03 399 S N
0.07 0.02 294 S T 0.23 0.06 402 R K 0.11 0.06 297 T M 0.18 0.07 403
T L 0.09 0.04 304 E Q -0.02 0.03 404 D I -0.02 0.01 308 S R 0.05
0.03 404 D M 0.10 0.07 310 L R 0.26 0.07 404 D Q 0.35 0.07 333 L M
0.14 0.09 404 D S 0.27 0.07 336 Q M 0.02 0.05 408 N H -0.03 0.03
354 A H 0.12 0.03 409 S N -0.07 0.08 357 C V 0.31 0.06 441 N R 0.02
0.08 358 L F 0.08 0.04 448 G W 0.09 0.05 359 D N 0.28 0.09 449 E A
0.04 0.05 377 L I 0.10 0.08 469 V T 0.02 0.03 423 V H 0.25 0.06 472
L M -0.06 0.07 426 P K 0.21 0.07 473 C Q 0.30 0.04 428 K R 0.04
0.04 484 R K 0.15 0.10 434 S A -0.06 0.09 507 T C 0.17 0.03 438 S A
0.08 0.05 523 G A 0.10 0.03 447 T A 0.20 0.05 527 I M 0.05 0.11 447
T C -0.01 0.04 528 Y K 0.80 0.08 447 T G 0.34 0.07 543 Y I 0.20
0.06 450 L V 0.08 0.05 549 E A 0.18 0.02 462 A H 0.67 0.03 550 K M
0.28 0.07 462 A Q 0.37 0.04 556 S V -0.04 0.07 467 V C -0.04 0.04
557 P S 0.22 0.06 469 I V 0.21 0.05 559 H K -0.04 0.06 472 I L 0.01
0.06 560 V F -0.01 0.02 476 L M -0.02 0.05 561 N P -0.04 0.05 488 P
E 0.00 0.05 562 V Y -0.08 0.05 498 Q M 0.17 0.09 567 V H 0.00 0.05
502 L V 0.31 0.07 567 V I 0.02 0.05 517 E I 0.05 0.02 583 S M -0.02
0.05 520 P D 0.35 0.05 601 E V 0.31 0.06 520 P G 0.09 0.07 605 E C
-0.11 0.09 520 P K 0.00 0.03 605 E H 0.28 0.05 521 S G 0.00 0.05
605 E M -0.06 0.06 523 N S 0.34 0.05 605 E W 0.05 0.05 533 I E 0.02
0.07 607 D C -0.05 0.02 534 D A 0.17 0.04 607 D H 0.04 0.03 576 F E
0.12 0.05 607 D K -0.02 0.01 576 F R 0.42 0.06 607 D N -0.02 0.04
577 K I 0.26 0.03 609 S H 0.25 0.03 582 I R 0.01 0.07 609 S V -0.02
0.01 583 Y F 0.06 0.07 610 L I 0.19 0.03 587 L W 0.03 0.07 587 L Y
0.35 0.06
TABLE-US-00012 TABLE 12 C G H I J K L M A B Transposase D E F
DNA:RNA Tran- Tran- Selec- Selec- Re- Re- 1 RNA DNA SEQ* Transposon
Transposon Transposase Ratio sient sient tion tion covery covery 2
no yes 48 CMV no insulators N/A none N/A 1150 629 172 157 233 143 3
yes no 48 CMV no insulators 750 ng 250 ng 3:1 735 976 351 916 4211
4229 4 yes no 48 CMV no insulators 660 ng 330 ng 2:1 516 509 228
184 1505 822 5 yes no 48 CMV no insulators 500 ng 500 ng 1:1 436
351 146 139 134 118 6 yes no 168 CMV no insulators 750 ng 250 ng
3:1 1006 476 1342 2053 4229 6040 7 yes no 168 CMV no insulators 660
ng 330 ng 2:1 842 770 1918 4350 5936 5709 8 yes no 168 CMV no
insulators 500 ng 500 ng 1:1 548 542 2263 1284 5162 4927 9 yes no
189 CMV no insulators 750 ng 250 ng 3:1 1107 420 2073 1072 5883
5323 10 yes no 189 CMV no insulators 660 ng 330 ng 2:1 837 654 1119
1796 5126 6111 11 yes no 189 CMV no insulators 500 ng 500 ng 1:1
664 680 3935 2853 6218 4647 12 yes no 175 CMV no insulators 750 ng
250 ng 3:1 872 468 3442 3012 5676 7511 13 yes no 175 CMV no
insulators 660 ng 330 ng 2:1 928 605 2479 2233 5616 5173 14 yes no
175 CMV no insulators 500 ng 500 ng 1:1 644 508 3832 1840 5276 5344
15 no no none none 0 0 N/A 236 280 143 140 143 122 *SEQ ID NO.
TABLE-US-00013 TABLE 13 F G A B C puro trans- trans- left right D E
promoter posase H I J 1 poson SEQ* SEQ* Int Seq system SEQ* SEQ*
GFP1 GFP2 GFP3 2 187151 2 12 5'-TTAA-3' Xenopus 937 175 875 63 979
3 187151 2 12 5'-TTAA-3' Xenopus 937 189 909 957 135 4 187151 2 12
5'-TTAA-3' Xenopus 937 none 236 84 84 5 241555 1095 11 5'-TTAA-3'
Xenopus 942 189 2594 91 3168 6 241555 1095 11 5'-TTAA-3' Xenopus
942 175 2934 3746 4365 7 241555 1095 11 5'-TTAA-3' Xenopus 942 none
94 93 102 8 246143 2 12 5'-TTAA-3' Xenopus 942 175 2445 2361 2324 9
246143 2 12 5'-TTAA-3' Xenopus 942 none 66 68 63 10 194094 23 29
5'-TTAT-3' Bombyx 937 407 426 710 630 11 194094 23 29 5'-TTAT-3'
Bombyx 937 1098 708 89 741 12 194094 23 29 5'-TTAT-3' Bombyx 937
none 88 92 94 13 240671 22 30 5'-TTAA-3' Bombyx 937 407 641 89 89
14 240671 22 30 5'-TTAA-3' Bombyx 937 1098 664 808 681 15 240671 22
30 5'-TTAA-3' Bombyx 937 none 379 94 94 16 none N/A N/A N/A none
N/A N/A 87 91 87 *SEQ ID NO.
TABLE-US-00014 TABLE 14 A B C D Xenopus SEQ* hyperactivity Bombyx
SEQ* hyperactivity 228 9 654 1.3 244 7 639 1.9 247 6 634 2.0 252 6
619 3 268 5 614 3 51 0.4 1097 4 64 80 596 4 56 126 595 4 57 122 588
5 124 25 557 7 52 414 518 11 58 116 517 11 54 127 508 12 73 58 491
15 71 64 488 15 65 79 457 31 63 91 449 35 62 95 417 94 61 99 416 97
59 112 415 107 168 15 414 122 189 13 413 130 175 15 412 164 118 22
211 10 216 9 *SEQ ID NO.
TABLE-US-00015 TABLE 15 E G H puro F L R K A B C pro- GFP insu-
insu- Bombyx trans- left right D moter pro- lator lator I J
Transposase L M N O P Q poson SEQ* SEQ* Int Seq SEQ* moter SEQ*
SEQ* RNA DNA SEQ* A600 A600 A600 GFP GFP GFP 1 194094 23 29
5'-TTAT-3' 937 CMV none none yes no 1098 0.44 0.34 0.42 1947 1547
1876 2 194094 23 29 5'-TTAT-3' 937 CMV none none no yes 415 0.34
0.27 0.32 1455 1240 1231 3 194094 23 29 5'-TTAT-3' 937 CMV none
none no yes 457 0.28 0.34 0.30 1107 1152 1213 4 194094 23 29
5'-TTAT-3' 937 CMV none none no yes 417 0.30 0.30 0.34 1061 950
1098 5 194094 23 29 5'-TTAT-3' 937 CMV none none no yes 412 0.31
0.33 0.35 860 1049 1143 6 194094 23 29 5'-TTAT-3' 937 CMV none none
no yes 416 0.32 0.29 0.35 1016 910 1004 7 194094 23 29 5'-TTAT-3'
937 CMV none none no yes 407 0.31 0.30 0.30 943 800 866 8 194094 23
29 5'-TTAT-3' 937 CMV none none no no N/A 0.02 0.04 0.04 150 171
167 9 240671 22 30 5'-TTAA-3' 937 CMV none none yes no 1098 0.48
0.44 0.26 2177 1757 1016 10 240671 22 30 5'-TTAA-3' 937 CMV none
none no yes 415 0.34 0.30 0.35 1525 1480 1514 11 240671 22 30
5'-TTAA-3' 937 CMV none none no yes 457 0.29 0.34 0.31 1257 1191
1144 12 240671 22 30 5'-TTAA-3' 937 CMV none none no yes 412 0.34
0.29 0.28 1001 1032 897 13 240671 22 30 5'-TTAA-3' 937 CMV none
none no yes 416 0.27 0.33 0.29 917 874 953 14 240671 22 30
5'-TTAA-3' 937 CMV none none no yes 407 0.32 0.26 0.27 1006 784 885
15 240671 22 30 5'-TTAA-3' 937 CMV none none no yes 417 0.27 0.25
0.23 800 859 777 16 240671 22 30 5'-TTAA-3' 937 CMV none none no no
N/A 0.03 0.17 0.06 178 261 168 17 246143 22 30 5'-TTAA-3' 942 CMV
none none no yes 415 0.00 0.00 0.00 102 109 142 18 246143 22 30
5'-TTAA-3' 942 CMV none none no yes 412 0.00 0.00 0.00 114 103 107
19 246143 22 30 5'-TTAA-3' 942 CMV none none no yes 416 0.00 -0.01
0.00 109 102 106 20 246143 22 30 5'-TTAA-3' 942 CMV none none no
yes 417 0.00 0.00 0.00 106 100 98 21 246143 22 30 5'-TTAA-3' 942
CMV none none no yes 407 0.00 -0.01 0.00 105 98 101 22 246143 22 30
5'-TTAA-3' 942 CMV none none yes no 1098 -0.01 0.00 0.00 99 104 96
23 246143 22 30 5'-TTAA-3' 942 CMV none none no yes 457 0.00 0.00
0.00 97 101 100 24 246143 22 30 5'-TTAA-3' 942 CMV none none no no
N/A 0.00 0.00 0.00 109 105 104 25 246170 22 30 5'-TTAA-3' 942 EF1a
864 860 no yes 415 0.18 0.00 0.04 5477 162 1559 26 246170 22 30
5'-TTAA-3' 942 EF1a 864 860 no yes 412 0.03 0.04 0.06 1148 1589
3145 27 246170 22 30 5'-TTAA-3' 942 EF1a 864 860 no yes 417 0.02
0.01 0.00 637 683 203 28 246170 22 30 5'-TTAA-3' 942 EF1a 864 860
no yes 416 0.00 0.02 0.00 146 652 217 29 246170 22 30 5'-TTAA-3'
942 EF1a 864 860 yes no 1098 0.00 0.01 0.00 237 286 118 30 246170
22 30 5'-TTAA-3' 942 EF1a 864 860 no yes 457 0.00 0.00 0.00 106 122
115 31 246170 22 30 5'-TTAA-3' 942 EF1a 864 860 no yes 407 0.00
0.00 0.00 108 101 113 32 246170 22 30 5'-TTAA-3' 942 EF1a 864 860
no no N/A 0.00 0.00 0.00 108 128 114 *SEQ ID NO.
TABLE-US-00016 TABLE 16 B D E F puro C L R Uribo A pro- GFP insu-
insu- Trans- trans- moter pro- lator lator posase G H I poson SEQ*
moter SEQ* SEQ* SEQ* GFP GFP GFP 1 246143 942 CMV none none 48 94
94 112 2 246143 942 CMV none none 58 99 2600 111 3 246143 942 CMV
none none none 107 94 98 4 246170 942 EF1a 864 860 48 95 93 108 5
246170 942 EF1a 864 860 61 4075 113 94 6 246170 942 EF1a 864 860
none 114 95 100 7 261961 948 EF1a 864 864 48 96 97 112 8 261961 948
EF1a 864 864 57 128 2008 490 9 261961 948 EF1a 864 864 none 86 104
94 *SEQ ID NO.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20220282260A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20220282260A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References