U.S. patent application number 11/569335 was filed with the patent office on 2009-07-02 for methods for dynamic vector assembly of dna cloning vector plasmids.
This patent application is currently assigned to INTREXON CORPORATION. Invention is credited to Thomas D. Reed.
Application Number | 20090170727 11/569335 |
Document ID | / |
Family ID | 35450900 |
Filed Date | 2009-07-02 |
United States Patent
Application |
20090170727 |
Kind Code |
A1 |
Reed; Thomas D. |
July 2, 2009 |
METHODS FOR DYNAMIC VECTOR ASSEMBLY OF DNA CLONING VECTOR
PLASMIDS
Abstract
A method for using cloning vector plasmids to produce DNA
molecules, such as transgenes, in a single cloning step. The
transgenes can be used for the purpose of gene expression or
analysis of gene expression. The plasmid cloning vectors are
engineered to minimize the amount of manipulation of DNA fragment
components by the end user of the vectors and the methods for their
use. Transgenes produced using the invention may be used in a
single organism, or in a variety of organisms including bacteria,
yeast, mice, and other eukaryotes with little or no further
modification.
Inventors: |
Reed; Thomas D.;
(Blacksburg, VA) |
Correspondence
Address: |
Sterne, Kessler, Goldstein & Fox P.L.L.C.
1100 New York Avene, N.W.
Washington
DC
20005
US
|
Assignee: |
INTREXON CORPORATION
Blacksburg
VA
|
Family ID: |
35450900 |
Appl. No.: |
11/569335 |
Filed: |
May 18, 2005 |
PCT Filed: |
May 18, 2005 |
PCT NO: |
PCT/US05/17272 |
371 Date: |
February 22, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60572011 |
May 18, 2004 |
|
|
|
Current U.S.
Class: |
506/26 ;
435/91.41 |
Current CPC
Class: |
C07H 21/04 20130101;
C12N 15/66 20130101; C12N 15/64 20130101; C12N 15/85 20130101 |
Class at
Publication: |
506/26 ;
435/91.41 |
International
Class: |
C40B 50/06 20060101
C40B050/06; C12N 15/66 20060101 C12N015/66 |
Claims
1. A method for constructing a transgene, comprising the steps of:
a. providing a cloning vector plasmid with a backbone able to
accept a sequential arrangement of inserts; b. providing at least a
first insert and a second insert to be included in the transgene;
and c. transferring both the first insert and the second insert
into the backbone in a single reaction.
2. A method for making a transgene, comprising the steps of: a.
providing a cloning vector plasmid comprising first and second
docking points; b. introducing first nucleotide sequences to be
included in the transgene into a first shuttle vector; c.
introducing second nucleotide sequences to be included in the
transgene into a second shuttle vector; and d. transferring
simultaneously the first nucleotide sequences and the second
nucleotide sequences from the shuttle vectors to the cloning vector
plasmid, between the first and second docking points.
3. A method for making a transgene, comprising the steps of: a.
providing a cloning vector plasmid comprising first and second
docking points; b. introducing Promoter nucleotide sequences to be
included in the transgene into a Promoter shuttle vector; c.
introducing Expression nucleotide sequences to be included in the
transgene into an Expression shuttle vector; d. introducing
Regulatory nucleotide sequences to be included in the transgene
into a Regulatory shuttle vector; and e. transferring
simultaneously the Promoter, Expression and Regulatory nucleotide
sequences from the Promoter, Expression and Regulatory shuttle
vectors to the cloning vector plasmid, between the first and second
docking points.
4. A method for simultaneously synthesizing an array of transgenes,
comprising the steps of: a. providing a primary cloning vector
plasmid comprising a first and a second docking point; b.
introducing at least one Promoter nucleotide sequence to be
included in the transgene into a corresponding Promoter shuttle
vector; c. introducing at least one Expression nucleotide sequence
to be included in the transgene into a corresponding Expression
shuttle vector; d. introducing at least one Regulatory nucleotide
sequence to be included in the transgene into a corresponding
Regulatory shuttle vector; and e. transferring simultaneously the
Promoter, Expression and Regulatory nucleotide sequences from the
Promoter, Expression and Regulatory shuttle vectors to the cloning
vector plasmid, between the first and second docking points; f.
wherein at least two combinations of one Promoter module, one
Expression module, and one Regulatory module are transferred into
two distinct primary cloning vector molecules.
5. A method for making a modular cloning vector plasmid for the
synthesis of a transgene or other complicated DNA construct, the
method comprising the steps of: a. providing the cloning vector
plasmid comprising a backbone, the backbone comprising first and
second docking points, each docking point being fixed within the
backbone and comprising at least one non-variable rare endonuclease
site for an endonuclease enzyme; b. cleaving the first docking
point with a first endonuclease enzyme corresponding to the at
least one non-variable rare restriction site of the first docking
point, leaving the cleaved first docking point with a 3' end; c.
cleaving the second docking point with a second nuclease enzyme
corresponding to the at least one non-variable rare endonuclease
site of the second docking point, leaving the cleaved second
docking point with a 5' end; d. providing at least a first and a
second insert, each insert comprising a 5' end, a nucleotide
sequence of interest and a 3' end, wherein the 5' end of the first
insert is compatible to the 3' end of the cleaved first docking
point, the 3' end of the second insert is compatible to the 5' end
of the cleaved second docking point, the 3' end of the first insert
being compatible to the 5' end of the second insert to form a third
non-variable rare endonuclease site for a third endonuclease
enzyme; and e. placing the inserts and the cleaved cloning vector
plasmid into an appropriate reaction mixture to cause simultaneous
ligation and self-orientation of the first and second inserts
between the first and second docking points within the backbone,
re-forming the first and second docking points, and forming the
modular cloning vector plasmid.
6. The method of claim 5 that provides for modifying the modular
cloning vector plasm further comprising the steps of: f.
subsequently removing the first insert by cleaving the modular
cloning vector plasmid at the first docking point and the third
non-variable rare endonuclease site with the first and the third
endonuclease enzymes, leaving a 3' end at the cleaved first docking
point and a 5' end at the cleaved third endonuclease site; g.
providing a third insert comprising a 5' end, a nucleotide sequence
of interest and a 3' end, wherein the 5' end of the third insert is
compatible to the 3' end of the cleaved first docking point', and
the 3' end of the third insert is compatible to the 5' end of the
cleaved third endonuclease site; and h. placing the third insert
and the cleaved modular cloning vector plasmid into an appropriate
reaction mixture to cause simultaneous ligation and
self-orientation of the third insert between the first docking
point and the third endonuclease site, and re-forming the first
docking point and the third endonuclease site.
7. The method of claim 5 that provides for modifying the modular
cloning vector plasm further comprising the steps of: i.
subsequently removing the second insert by cleaving the modular
cloning vector plasmid at the first docking point and the third
non-variable rare endonuclease site with the second and the third
endonuclease enzymes, leaving a 3' end at the cleaved third
endonuclease site and a 5' end at the cleaved second docking point;
j. providing a fourth insert comprising a 5' end, a nucleotide
sequence of interest and a 3' end, wherein the 5' end of the fourth
insert is compatible to the 3' end of the cleaved third
endonuclease site, and the 3' end of the fourth insert is
compatible to the 5' end of the cleaved second docking point; and
k. placing the fourth insert and the cleaved modular cloning vector
plasmid into an appropriate reaction mixture to cause simultaneous
ligation and self-orientation of the fourth insert between the
third endonuclease site and the second docking point, and
re-forming the third endonuclease site and the second docking
point.
8. The method of claim 5, wherein the inserts are created by a
method selected from the group consisting of de novo synthesis,
recombineering, and PCR terminator over-hang cloning.
9. A method for synthesizing a transgene or other complicated DNA
construct, comprising the steps of: a. providing a primary cloning
vector plasmid comprising a backbone, the backbone comprising at
least a first docking point and a second docking point, each
docking point being fixed within the backbone and comprising at
least one rare restriction site for a non-variable rare restriction
enzyme; b. cleaving the first docking point with a first
non-variable rare restriction enzyme corresponding to one of the
rare restriction sites of the first docking point, leaving the
cleaved backbone with a 3' end; c. cleaving the second docking
point with a second non-variable rare restriction enzyme
corresponding to one of the restriction sites of the second docking
point, leaving the cleaved backbone with a 5' end; d. providing a
Promoter insert into which a Promoter sequence of interest, a 5'
end that is compatible to the 3' end of the first docking point,
and a 3' end; e. providing an Expression insert comprising an
Expression sequence of interest, a 5' end that is compatible to the
3' end of the Promoter insert to form a rare restriction site for a
third non-variable rare restriction enzyme, and a 3' end; f.
providing a Regulatory insert comprising a Regulatory sequence of
interest, a 5' end that is compatible to the 3' end of the
Expression insert to form a rare restriction site for a fourth
non-variable rare restriction enzyme, and a 3' end that is
compatible to the 5' end of the cleaved second docking point which
was cleaved in step `c`; and g. placing the Promoter, Expression
and Regulatory inserts and the cleaved cloning vector plasmid into
an appropriate reaction mixture to cause simultaneous ligation,
self-orientation and sequential placement of the Promoter,
Expression and Regulatory inserts between the first and second
docking points, reforming the first and second docking points, and
forming a modular primary cloning vector plasmid.
10. The method of claim 9 that provides for modifying the modular
primary cloning vector plasmid, further comprising the steps of: h.
cleaving the modular primary cloning vector plasmid with a pair of
rare restriction enzymes at a corresponding pair of rare
restriction sites of at least one of the Promoter, Expression and
Regulatory inserts, leaving the cleaved vector plasmid with a 3'
end and a 5' end; i. providing at least one fifth insert selected
from a Promoter insert, an Expression insert, and a Regulatory
insert, the fifth insert having a 5' end that is compatible to the
3' end of the cleaved vector plasmid, and a 3' end that is
compatible to the 5' end of the cleaved vector plasmid; and j.
placing the fifth insert and the cleaved vector plasmid into an
appropriate reaction mixture to cause simultaneous ligation and
self-orientation of the fifth insert into the modular primary
cloning vector plasmid, in the same sequence, and re-forming the
pair of rare restriction sites.
11. The method of claim 10, further comprising the step of
repeating steps h, i, and j for one or more additional inserts
selected from a Promoter insert, an Expression insert, and a
Regulatory insert.
12. A method for simultaneously synthesizing an array of transgenes
or other complicated DNA constructs, comprising the steps of: a.
providing at least one primary cloning vector plasmid comprising a
backbone into which inserts having a 5' end, a nucleotide sequence
of interest and a 3' end can be inserted, the backbone operable to
accept a sequential arrangement of Promoter, Expression, and
Regulatory inserts and comprising at least a first and a second
docking point, each docking point being fixed within the backbone
and comprising at least one restriction site for a non-variable
rare restriction enzyme; b. cleaving the first docking point with a
first non-variable rare restriction enzyme corresponding to one of
the restriction sites of the first docking point; c. cleaving the
second docking point with a second non-variable rare restriction
enzyme corresponding to one of the restriction sites of the second
docking point; d. providing at least one Promoter insert into which
a Promoter nucleotide sequence has been inserted, the 5' end of the
at least one Promoter insert compatible to the 3' end of the first
docking point which was cleaved in step `b`; e. providing at least
one Expression insert into which an Expression nucleotide sequence
has been inserted, the 5' end of the at least one Expression insert
being compatible to the 3' end of the at least one Promoter insert
to form a restriction site for a third non-variable rare
restriction enzyme; f. providing at least one Regulatory insert
into which a Regulatory nucleotide sequence has been inserted, the
5' end of the at least one Regulatory insert being compatible to
the 3' end of the at least one Expression insert to form a
restriction site for a fourth non-variable rare restriction enzyme,
the 3' end of the at least one Regulatory insert compatible to the
5' end of the of the second docking point which was cleaved in step
`c`; and g. thereafter placing at least two different types of at
least one of the Promoter, Expression and Regulatory inserts, at
least one of each of the remaining inserts, and the cleaved cloning
vector plasmid into an appropriate reaction mixture to cause
simultaneous ligation, self-orientation and sequential placement of
one each of the Promoter, Expression and Regulatory inserts between
the first and second docking points within the backbone, thereby
creating an array of plasmids having different combinations of
Promoter, Expression and Regulatory inserts within their
backbone.
13. The method of claim 12, wherein step `g` comprises placing at
least two different types of at least two of the Promoter,
Expression and Regulatory inserts, at least one of each of the
remaining inserts, and the cleaved cloning vector plasmid into an
appropriate reaction mixture to cause simultaneous ligation,
self-orientation and sequential placement of one each of the
Promoter, Expression, and Regulatory inserts between the first and
second docking points within the backbone, thereby creating an
array of plasmids having different combinations of Promoter,
Expression and Regulatory inserts within their backbone.
14. The method of claim 12, wherein step `g` comprises placing at
least two different types of each of the Promoter, Expression and
Regulatory inserts and the cleaved cloning vector plasmid into an
appropriate reaction mixture to cause simultaneous ligation,
self-orientation and sequential placement of one each of the
Promoter, Expression, and Regulatory inserts between the first and
second docking points within the backbone, thereby creating an
array of plasmids having different combinations of Promoter,
Expression and Regulatory inserts within their backbone.
15. The method of claims 12, 13, or 14 that provides for modifying
the modular primary cloning vector plasmid, further comprising the
step of: h. assaying the products of step `g` using high throughput
screening; i. isolating a specific product of step `g` which is a
primary cloning vector plasmid; j. cleaving the backbone of primary
cloning vector plasmid from step `i` at the 5' and 3' ends of one
of the Promoter, Expression and Regulatory inserts with the pair of
rare restriction enzymes corresponding thereto; k. providing a
sixth insert, the 5' end of the sixth insert compatible to the 3'
end of the backbone which was cleaved in step `j`, the 3' end of
the sixth insert compatible to the 5' end of the backbone which was
cleaved in step `j`; and l. thereafter placing the sixth insert and
the backbone which was cleaved in step `j` into an appropriate
reaction mixture to cause simultaneous ligation and
self-orientation of the sixth insert into the backbone.
16. The method of claim 13, further comprising the step of
repeating steps h, i, j, k and I for any number of inserts in order
to sequentially replace one insert for another insert into the
backbone.
17. The method of claim 12, wherein the inserts are created by a
method selected from the group consisting of de novo synthesis,
recombineering, PCR, terminator over-hang cloning technology, and
restriction endonuclease mapping.
18. The method of any of claims 5, 9 or 12, wherein the backbone
further comprises a unique HE site in a forward orientation located
upstream from the 5' end of the first docking point and a unique HE
site in a reverse orientation located downstream from the 3' end of
the second docking point, the method further comprising the steps
of: m. cleaving the backbone at each of the unique HE sites with a
unique HE restriction enzyme; n. purifying the cleaved portion
containing the inserts; and o. inserting the cleaved portion into a
genome host of interest.
Description
BACKGROUND OF THE INVENTION
[0001] The present invention relates in general to the field of
cloning vector plasmids, and in particular to methods for rapidly
assembling DNA constructs or transgenes with cloning vector
plasmids.
[0002] The foundation of molecular biology is recombinant DNA
technology, which can here be summarized as the modification and
propagation of nucleic acids for the purpose of studying the
structure and function of the nucleic acids and their protein
products.
[0003] Individual genes, gene regulatory regions, subsets of genes,
and indeed entire chromosomes in which they are contained, are all
comprised of double-stranded anti-parallel sequences of the
nucleotides adenine, thymine, guanine and cytosine, identified
conventionally by the initials A, T, G, and C, respectively. These
DNA sequences, as well as cDNA sequences, which are double stranded
DNA copies derived from mRNA (messenger RNA) molecules, can be
cleaved into distinct fragments, isolated, and inserted into a
vector such as a bacterial plasmid to study the gene products. A
plasmid is an extra-chromosomal piece of DNA that was originally
derived from bacteria, and can be manipulated and reintroduced into
a host bacterium for the purpose of study or production of a gene
product. The DNA of a plasmid is similar to all chromosomal DNA, in
that it is composed of the same A, T, G, and C nucleotides encoding
genes and gene regulatory regions, however, it is a relatively
small molecule comprised of less than approximately 30,000
base-pairs, or 30 kilobases (kb). In addition, the nucleotide base
pairs of a double-stranded plasmid form a continuous circular
molecule, also distinguishing plasmid DNA from that of chromosomal
DNA.
[0004] Plasmids enhance the rapid exchange of genetic material
between bacterial organisms and allow rapid adaptation to changes
in environment, such as temperature, food supply, or other
challenges. Any plasmid acquired must express a gene or genes that
contribute to the survival of the host or else it will be destroyed
or discarded by the organism, since the maintenance of unnecessary
plasmids would be a wasteful use of resources. A clonal population
of cells contains identical genetic material, including any
plasmids it might harbor. Use of a cloning vector plasmid with a
DNA insert in such a clonal population of host cells will amplify
the amount of the DNA of interest available. The DNA so cloned may
then be isolated and recovered for subsequent manipulation in the
steps required for building a DNA construct. Thus, it can be
appreciated that cloning vector plasmids are useful tools in the
study of gene function, providing the ability to rapidly produce
large amounts of the DNA insert of interest.
[0005] While some elements found in plasmids are naturally
occurring, others have been engineered to enhance the usefulness of
plasmids as DNA vectors. These include antibiotic- or
chemical-resistance genes and a multiple cloning site (MCS), among
others. Each of these elements has a role in the present invention,
as well as in the prior art. Description of the role each element
plays will highlight the limitations of the prior art and
demonstrate the utility of the present invention.
[0006] A particularly useful plasmid-born gene that can be acquired
by a host is one that would confer antibiotic resistance. In the
daily practice of recombinant DNA technology, antibiotic resistance
genes are exploited as positive or negative selection elements to
preferentially enhance the culture and amplification of the desired
plasmid over that of other plasmids.
[0007] In order to be maintained by a host bacterium, a plasmid
must also contain a segment of sequences that direct the host to
duplicate the plasmid. Sequences known as the origin of replication
(ORI) element direct the host to use its cellular enzymes to make
copies of the plasmid. When such a bacterium divides, the daughter
cells will each retain a copy or copies of any such plasmid.
Certain strains of E. coli bacteria have been derived to maximize
this duplication, producing upwards of 300 copies per bacterium. In
this manner, the cultivation of a desired plasmid can be
enhanced.
[0008] Another essential element in any cloning vector is a
location for insertion of the genetic materials of interest. This
is a synthetic element that has been engineered into "wild type"
plasmids, thus conferring utility as a cloning vector. Any typical
commercially-available cloning vector plasmid contains at least one
such region, known as a multiple cloning site (MCS). A MCS
typically comprises nucleotide sequences that are cleaved by a
single endonuclease enzyme, or a series of endonuclease enzymes (,
each of which has a distinct recognition sequence and cleavage
pattern. The so-called recognition sequences of a restriction
endonuclease (RE) site encoded in the DNA molecule comprise
double-stranded palindromic sequences. For some RE enzymes, as few
as 4-6 nucleotides are sufficient to provide a recognition site,
while some RE enzymes require a sequence of 8 or more nucleotides.
The RE enzyme EcoR1, for example, recognizes the double-stranded
hexanucleotide sequence: .sup.5' G-A-A-T-T-C.sup.3', wherein 5'
indicates the end of the molecule known by convention as the
"upstream" end, and 3' likewise indicates the "downstream" end. The
complementary strand of the recognition sequence would be its
anti-parallel strand, .sup.3' G-A-A-T-T-C-.sup.5'. Since every
endonuclease site is a double-stranded sequence of nucleotides, a
recognition site of 6 nucleotides is, in fact, 6 base pairs (bp).
Thus the double stranded recognition site can be represented within
the larger double-stranded molecule in which it occurs as:
TABLE-US-00001 .sup.5'......G-A-A-T-T-C.......sup.3' .sup.3
'......C-T-T-A-A-G.......sup.5'.
[0009] Like many other RE enzymes, EcoR1 does not cleave exactly at
the axis of dyad symmetry, but at positions four nucleotides apart
in the two DNA strands between the nucleotides indicated by a
"/":
TABLE-US-00002 .sup.5'......G/A-A-T-T-C........sup.3'
.sup.3'......C-T-T-A-A/G........sup.5',
[0010] such that double-stranded DNA molecule is cleaved and has
the resultant configuration of nucleotides at the newly formed
"ends":
TABLE-US-00003 .sup.5'......G .sup.3' 5' A-A-T-T-C........sup.3'
.sup.3'......C-T-T-A-A .sup.5' 3' G........sup.5'
[0011] This staggered cleavage yields fragments of DNA with
protruding 5' termini. Because A-T and G-C pairs are spontaneously
formed when in proximity with each other, protruding ends such as
these are called cohesive or sticky ends. Any one of these termini
can form hydrogen bonds with any other complementary termini
cleaved with the same restriction enzyme. Since any DNA that
contains a specific recognition sequence will be cut in the same
manner as any other DNA containing the same sequence, those cleaved
ends will be complementary. Therefore, the ends of any DNA
molecules cut with the same RE enzyme "match" each other in the way
adjacent pieces of a jigsaw puzzle "match", and can be
enzymatically linked together. It is this property that permits the
formation of recombinant DNA molecules, and allows the introduction
of foreign DNA fragments into bacterial plasmids, or into any other
DNA molecule.
[0012] A further general principle to consider when building
recombinant DNA molecules is that all endonuclease sites occurring
within a molecule will be cut with a particular RE enzyme, not just
the site of interest. The larger a DNA molecule, the more likely it
is that any endonuclease site will reoccur. Assuming that any
endonuclease sites are distributed randomly along a DNA molecule, a
tetranucleotide site will occur, on the average, once every 4.sup.4
(i.e., 256) nucleotides or bp, whereas a hexanucleotide site will
occur once every 4.sup.6 (i.e., 4096) nucleotides or bp, and
octanucleotide sites will occur once every 4.sup.8 (i.e., 114,688)
nucleotides or bp. Thus, it can be readily appreciated that shorter
recognition sequences will occur frequently, while longer ones will
occur rarely. When planning the construction of a transgene or
other recombinant DNA molecule, this is a vital issue, since such a
project frequently requires the assembly of several pieces of DNA
of varying sizes. The larger these pieces are, the more likely that
the sites one wishes to use occur in several pieces of the DNA
components, making manipulation difficult at best.
[0013] Frequently-occurring endonuclease enzyme sites are herein
referred to as common sites, and the endonucleases that cleave
these sites are referred to as common endonuclease enzymes.
Restriction enzymes with cognate restriction sites greater than 6
bp are referred to as rare restriction enzymes, and their cognate
restriction sites as rare restriction sites. However, there are
some endonuclease sites of 6 bp that occur more infrequently than
would be statistically predicted, and these sites and the
endonucleases that cleave them are also referred to as rare. Thus,
the designations "rare" and common" do not refer to the relative
abundance or availability of any particular restriction enzyme, but
rather to the frequency of occurrence of the sequence of
nucleotides that make up its cognate recognition site within any
DNA molecule or isolated fragment of a DNA molecule, or any gene or
its DNA sequence.
[0014] A second class of endonuclease enzymes has recently been
isolated, called homing endonuclease (HE) enzymes. HE enzymes have
large, non-palindromic asymmetric recognition sites (12-40 base
pairs). HE recognition sites are extremely rare. For example, the
HE known as I-SceI has an 18 bp recognition site, (5' . . .
TAGGGATAACAGGGTAAT . . . 3'), predicted to occur only once in every
7.times.10.sup.10 bp of random sequence. This rate of occurrence is
equivalent to only one site in 20 mammalian-sized genomes. The rare
nature of HE recognition sites greatly increases the likelihood
that a genetic engineer can cut a final transgene product without
disrupting the integrity of the transgene if HE recognition sites
were included in appropriate locations in a cloning vector
plasmid.
[0015] Since a DNA molecule from any source organism will be cut in
identical fashion by an endonuclease enzyme, foreign pieces of DNA
from any species can be cut with an endonuclease enzyme, inserted
into a bacterial plasmid vector that was cleaved with the same
endonuclease enzyme, and amplified in a suitable host cell. For
example, if a human gene can cut in 2 places with the RE enzyme
known as EcoR1, the desired fragment with EcoR1 ends can be
isolated and mixed with a plasmid that was also cut with EcoR1 in
what is commonly known as a ligation mixture. Under the appropriate
conditions in the ligation mixture, some of the isolated human gene
fragments will match up with the ends of the plasmid molecules.
These newly joined ends can link together (ligated) to
enzymatically recircularize the plasmid, now containing its new DNA
insert. The ligation mixture is then introduced into E. coli or
another suitable host, and the newly engineered plasmids will be
amplified as the bacteria divide. In this manner, a relatively
large number of copies of the human gene may be obtained and
harvested from the bacteria. These gene copies can then be further
manipulated for the purpose of research, analysis, or production of
its gene product protein.
[0016] Recombinant DNA technology is frequently embodied in the
generation of so-called "transgenes". Transgenes frequently
comprise a variety of genetic materials that are derived from one
or more donor organisms and introduced into a host organism.
Typically, a transgene is constructed using a cloning vector as the
starting point or "backbone" of the project, and a series of
complex cloning steps are planned to assemble the final product
within that vector. Elements of a transgene, comprising nucleotide
sequences, include, but are not limited to 1) regulatory promoter
and/or enhancer elements, 2) a gene that will be expressed as a
mRNA molecule, 3) DNA elements that provide mRNA message
stabilization, 4) nucleotide sequences mimicking mammalian intronic
gene regions, and 5) signals for mRNA processing such as the poly-A
tail added to the end of naturally-occurring mRNAs. In some cases,
an experimental design may require addition of localization signal
to provide for transport of the gene product to a particular
subcellular location.
[0017] Each of the elements of a transgene can be derived as a
fragment of a larger DNA molecule that is cut from a donor genome,
or, in some cases, synthesized in a laboratory. While the present
invention employs endonucleases for the methods claimed herein, it
is known that each of the smaller elements comprising, for example,
the inserts or modules which are used in the methods herein, can be
created by de novo synthesis, recombineering, and/or PCR terminator
overhang cloning. One such method of synthesis of the component
elements of a transgene includes the method disclosed by Jarrell et
al. in U.S. Pat. No. 6,358,712, which is incorporated herein by
reference in its entirety. While Jarrell discloses a method for
"welding" elements of a transgene together, only the methods of the
present invention disclose a way to "unweld" and re-assemble the
elements once they have been assembled. According to one aspect of
the invention, each piece is assembled with the others in a precise
order and 5'-3' orientation into a cloning vector plasmid.
[0018] The promoter of any gene may be isolated as a DNA fragment
and placed within a synthetic molecule, such as a plasmid, to
direct the expression of a desired gene, assuming that the
necessary conditions for stimulation of the promoter of interest
can be provided. For example, the promoter sequences of the insulin
gene may be isolated, placed in a cloning vector plasmid along with
a reporter gene, and used to study the conditions required for
expression of the insulin gene in an appropriate cell type.
Alternatively, the insulin gene promoter may be joined with the
protein coding-sequence of any gene of interest in a cloning vector
plasmid, and used to drive expression of the gene of interest in
insulin-expressing cells, assuming that all necessary elements are
present within the DNA transgene so constructed.
[0019] A reporter gene is a particularly useful component of some
types of transgenes. A reporter gene comprises nucleotide sequences
encoding a protein that will be expressed under the direction of a
particular promoter of interest to which it is linked in a
transgene, providing a measurable biochemical response of the
promoter activity. A reporter gene is typically easy to detect or
measure against the background of endogenous cellular proteins.
Commonly used reporter genes include but are not limited to LacZ,
green fluorescent protein, and luciferase, and other reporter
genes, many of which are well known to those skilled in the
art.
[0020] Introns, which are non-coding regions within mammalian
genes, are not found in bacterial genomes, but are required for
proper formation of mRNA molecules in mammalian cells. Therefore,
any DNA construct for use in mammalian systems must have at least
one intron. Introns may be isolated from any mammalian gene and
inserted into a DNA construct, along with the appropriate splicing
signals that allow mammalian cells to excise the intron and splice
the remaining mRNA ends together.
[0021] An mRNA stabilization element is a sequence of DNA that is
recognized by binding proteins that protect some mRNAs from
degradation. Inclusion of an mRNA stabilization element will
frequently enhance the level of gene expression from that mRNA in
some mammalian cell types, and so can be useful in some DNA
constructs or transgenes. An mRNA stabilization element can be
isolated from naturally occurring DNA or RNA, or synthetically
produced for inclusion in a DNA construct.
[0022] A localization signal is a sequence of DNA that encodes a
protein signal for subcellular routing of a protein of interest.
For example, a nuclear localization signal will direct a protein to
the nucleus; a plasma membrane localization signal will direct it
to the plasma membrane, etc. Thus, a localization signal may be
incorporated into a DNA construct to promote the translocation of
its protein product to the desired subcellular location.
[0023] A tag sequence may be encoded in a DNA construct so that the
protein product will have a unique region attached. This unique
region serves as a protein tag that can distinguish it from its
endogenous counterpart. Alternatively, it can serve as an
identifier that may be detected by a wide variety of techniques
well known in the art, including, but not limited to, RT-PCR,
immunohistochemistry, or in situ hybridization.
[0024] With a complex transgene, or with one that includes
particularly large regions of DNA, there is an increased likelihood
that there will be multiple endonuclease recognition sites in these
pieces of DNA. Recall that the recognition sequences encoding any
one hexanucleotide site occur every 4096 bp (46). If a promoter
sequence is 3000 bp and a gene of interest of 1500 bp are to be
assembled into a cloning vector of 3000 bp, it is statistically
very likely that many sites of 6 or less nucleotides will not be
useful, since any usable sites must occur in only two of the
pieces. Furthermore, the sites must occur in the appropriate areas
of the appropriate molecules that are to be assembled. In addition,
most cloning projects will need to have additional DNA elements
added, thereby increasing the complexity of the growing molecule
and the likelihood of inopportune repetition of any particular
restriction site. Since any restriction enzyme will cut at all of
its sites in a molecule, if an endonuclease enzyme restriction site
reoccurs, all the inopportune sites will be cut along with the
desired sites, disrupting the integrity of the molecule. Thus, each
cloning step must be carefully planned so as not to disrupt the
growing molecule by cutting it with an endonuclease enzyme that has
already been used to incorporate a preceding element. And finally,
when a researcher wishes to introduce a completed transgene into a
mammalian organism, the fully-assembled transgene construct
frequently must be linearized at a unique recognition site at least
one end of the transgene, thus requiring yet another unique
recognition site found nowhere else in the construct. Since most
DNA constructs are designed for a single purpose, little thought is
given to any future modifications that might need to be made,
further increasing the difficulty for future experimental
changes.
[0025] Traditionally, transgene design and construction consumes
significant amounts of time and energy for several reasons,
including the following:
[0026] 1. There is a wide variety of endonuclease enzymes available
that will generate an array of termini, however most of these are
not compatible with each other. Many endonuclease enzymes, such as
EcoR1, generate DNA fragments with protruding 5' cohesive termini
or "tails"; others (e.g., Pst1) generate fragments with 3'
protruding tails, whereas still others (e.g., Bal1) cleave at the
axis of symmetry to produce blunt-ended fragments. Some of these
will be compatible with the termini formed by cleavage with other
endonuclease enzymes, but the majority of useful ones will not. The
termini that can be generated with each DNA fragment isolation must
be carefully considered in designing a DNA construct.
[0027] 2. DNA fragments needed for assembly of a DNA construct or
transgene must first be isolated from their source genomes, placed
into plasmid cloning vectors, and amplified to obtain useful
quantities. The step can be performed using any number of
commercially-available or individually altered cloning vectors.
Each of the different commercially available cloning vector
plasmids were, for the most part, developed independently, and thus
contain different sequences and endonuclease sites for the DNA
fragments of genes or genetic elements of interest. Genes must
therefore be individually tailored to adapt to each of these
vectors as needed for any given set of experiments. The same DNA
fragments frequently will need to be altered further for subsequent
experiments or cloning into other combinations for new DNA
constructs or transgenes. Since each DNA construct or transgene is
custom made for a particular application with no thought or
knowledge of how it will be used next, it frequently must be
"retrofitted" for subsequent applications.
[0028] 3. In addition, the DNA sequence of any given gene or
genetic element varies and can contain internal endonuclease sites
that make it incompatible with currently available vectors, thereby
complicating manipulation. This is especially true when assembling
several DNA fragments into a single DNA construct or transgene.
[0029] Thus, there remains a need for a system that would allow the
user to rapidly assemble a number of DNA fragments into one
molecule, despite redundancy of endonuclease sites found at the
ends and within the DNA fragments. Such a system might also provide
a simple means for rapidly altering the ends of the fragments so
that other endonuclease sequences are added to them. Inclusion of
single or opposing pairs of HE sites would enhance the likelihood
of having unique sites for cloning. A system that would also allow
easy substitutions or removal of one or more of the fragments would
add a level of versatility not currently available to users.
Therefore, a "modular" system, i.e. a system allowing one to insert
or remove DNA fragments or "inserts" into or out of "cassette"
regions flanked by rare endonuclease sites within the cloning
vector, would be especially useful and welcome to the field of
recombinant DNA technology.
SUMMARY OF THE INVENTION
[0030] Accordingly, the present invention provides a method of
rapidly assembling DNA constructs or transgenes by using cloning
vector plasmids. The invention also provides a method that
incorporates multiple DNA fragments, also known as both "inserts"
or "modules", such as one each of a Promoter, Expression, and 3'
Regulatory nucleotide sequence, into a cloning vector plasmid in a
single step, rather than having to introduce each insert in a
sequential manner. Such a method is called "Dynamic Vector
Assembly" herein.
[0031] In one embodiment, the present invention provides a method
for constructing a transgene, comprising the steps of providing a
cloning vector plasmid with a backbone able to accept a sequential
arrangement of inserts, providing at least a first insert and a
second insert to be included in the transgene, and transferring
both the first insert and the second insert into the backbone in a
single reaction.
[0032] In another embodiment, the invention provides a method for
making a transgene, comprising the steps of: providing a cloning
vector plasmid comprising first and second docking points;
introducing first nucleotide sequences to be included in the
transgene into a first shuttle vector; introducing second
nucleotide sequences to be included in the transgene into a second
shuttle vector; and transferring simultaneously the first
nucleotide sequences and the second nucleotide sequences from the
shuttle vectors to the cloning vector plasmid, between the first
and second docking points.
[0033] The invention also provides a method for making a transgene,
comprising the steps of: providing a cloning vector plasmid
comprising first and second docking points; introducing Promoter
nucleotide sequences to be included in the transgene into a
Promoter shuttle vector; introducing Expression nucleotide
sequences to be included in the transgene into an Expression
shuttle vector; introducing Regulatory nucleotide sequences to be
included in the transgene into a Regulatory shuttle vector; and
transferring simultaneously the Promoter, Expression and Regulatory
nucleotide sequences from the Promoter, Expression and Regulatory
shuttle vectors to the cloning vector plasmid, between the first
and second docking points.
[0034] In another embodiment, the invention provides a method for
simultaneously synthesizing an array of transgenes, comprising the
steps of: providing a primary cloning vector plasmid comprising a
first and a second docking point; introducing at least one Promoter
nucleotide sequence to be included in the transgene into a
corresponding Promoter shuttle vector; introducing at least one
Expression nucleotide sequence to be included in the transgene into
a corresponding Expression shuttle vector; introducing at least one
Regulatory nucleotide sequence to be included in the transgene into
a corresponding Regulatory shuttle vector; and transferring
simultaneously the Promoter, Expression and Regulatory nucleotide
sequences from the Promoter, Expression and Regulatory shuttle
vectors to the cloning vector plasmid, between the first and second
docking points, wherein at least two combinations of one Promoter
module, one Expression module, and one Regulatory module are
transferred into two distinct primary cloning vector molecules.
[0035] In yet another embodiment, the invention provides a method
for making a modular cloning vector plasmid for the synthesis of a
transgene or other complicated DNA construct, the method comprising
the steps of: providing the cloning vector plasmid comprising a
backbone, the backbone comprising first and second docking points,
each docking point being fixed within the backbone and comprising
at least one non-variable rare endonuclease site for an
endonuclease enzyme; cleaving the first docking point with a first
endonuclease enzyme corresponding to the at least one non-variable
rare restriction site of the first docking point, leaving the
cleaved first docking point with a 3' end; cleaving the second
docking point with a second nuclease enzyme corresponding to the at
least one non-variable rare endonuclease site of the second docking
point, leaving the cleaved second docking point with a 5' end;
providing at least a first and a second insert, each insert
comprising a 5' end, a nucleotide sequence of interest and a 3'
end, wherein the 5' end of the first insert is compatible to the 3'
end of the cleaved first docking point, the 3' end of the second
insert is compatible to the 5' end of the cleaved second docking
point, the 3' end of the first insert being compatible to the 5'
end of the second insert to form a third non-variable rare
endonuclease site for a third endonuclease enzyme; and placing the
inserts and the cleaved cloning vector plasmid into an appropriate
reaction mixture to cause simultaneous ligation and
self-orientation of the first and second inserts between the first
and second docking points within the backbone, re-forming the first
and second docking points, and forming the modular cloning vector
plasmid.
[0036] In another embodiment, the invention provides a method for
synthesizing a transgene or other complicated DNA construct,
comprising the steps of: providing a primary cloning vector plasmid
comprising a backbone, the backbone comprising at least a first
docking point and a second docking point, each docking point being
fixed within the backbone and comprising at least one rare
restriction site for a non-variable rare restriction enzyme;
cleaving the first docking point with a first non-variable rare
restriction enzyme corresponding to one of the rare restriction
sites of the first docking point, leaving the cleaved backbone with
a 3' end; cleaving the second docking point with a second
non-variable rare restriction enzyme corresponding to one of the
restriction sites of the second docking point, leaving the cleaved
backbone with a 5' end, providing a Promoter insert into which a
Promoter sequence of interest, a 5' end that is compatible to the
3' end of the first docking point, and a 3' end; providing an
Expression insert comprising an Expression sequence of interest, a
5' end that is compatible to the 3' end of the Promoter insert to
form a rare restriction site for a third non-variable rare
restriction enzyme, and a 3' end; providing a Regulatory insert
comprising a Regulatory sequence of interest, a 5' end that is
compatible to the 3' end of the Expression insert to form a rare
restriction site for a fourth non-variable rare restriction enzyme,
and a 3' end that is compatible to the 5' end of the cleaved second
docking point which was cleaved in step `c`; and placing the
Promoter, Expression and Regulatory inserts and the cleaved cloning
vector plasmid into an appropriate reaction mixture to cause
simultaneous ligation, self-orientation and sequential placement of
the Promoter, Expression and Regulatory inserts between the first
and second docking points, reforming the first and second docking
points, and forming a modular primary cloning vector plasmid.
[0037] In yet another embodiment, the invention provides a method
for simultaneously synthesizing an array of transgenes or other
complicated DNA constructs, comprising the steps of: providing at
least one primary cloning vector plasmid comprising a backbone into
which inserts having a 5' end, a nucleotide sequence of interest
and a 3' end can be inserted, the backbone operable to accept a
sequential arrangement of Promoter, Expression, and Regulatory
inserts and comprising at least a first and a second docking point,
each docking point being fixed within the backbone and comprising
at least one restriction site for a non-variable rare restriction
enzyme; cleaving the first docking point with a first non-variable
rare restriction enzyme corresponding to one of the restriction
sites of the first docking point; cleaving the second docking point
with a second non-variable rare restriction enzyme corresponding to
one of the restriction sites of the second docking point; providing
at least one Promoter insert into which a Promoter nucleotide
sequence has been inserted, the 5' end of the at least one Promoter
insert compatible to the 3' end of the first docking point which
was cleaved in step `b`; providing at least one Expression insert
into which an Expression nucleotide sequence has been inserted, the
5' end of the at least one Expression insert being compatible to
the 3' end of the at least one Promoter insert to form a
restriction site for a third non-variable rare restriction enzyme;
providing at least one Regulatory insert into which a Regulatory
nucleotide sequence has been inserted, the 5' end of the at least
one Regulatory insert being compatible to the 3' end of the at
least one Expression insert to form a restriction site for a fourth
non-variable rare restriction enzyme, the 3' end of the at least
one Regulatory insert compatible to the 5' end of the of the second
docking point which was cleaved in step `c`; and thereafter placing
at least two different types of at least one of the Promoter,
Expression and Regulatory inserts, at least one of each of the
remaining inserts, and the cleaved cloning vector plasmid into an
appropriate reaction mixture to cause simultaneous ligation,
self-orientation and sequential placement of one each of the
Promoter, Expression and Regulatory inserts between the first and
second docking points within the backbone, thereby creating an
array of plasmids having different combinations of Promoter,
Expression and Regulatory inserts within their backbone.
[0038] A further understanding of the nature and advantages of the
present invention will be more fully appreciated with respect to
the following drawings and detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0039] The accompanying drawings, which are incorporated in and
constitute a part of this specification, illustrate embodiments of
the invention and, together with a general description of the
invention given above, and the detailed description given below,
serve to explain the principles of the invention.
[0040] FIG. 1 is a linear map of the module concept of the
invention showing a Shuttle vector that is insertable into a PE3
docking station, which is insertable into a Primary docking
station.
[0041] FIG. 2 is an illustration depicting assembly of a backbone
vector enabled by the relationships between restriction sites
within shuttle vectors such as Promoter, Expression and 3'
Regulatory modules, and the docking points on a primary cloning
vector plasmid.
[0042] FIG. 3 is an illustration depicting assembled backbone
vector of FIG. 2.
DETAILED DESCRIPTION OF THE INVENTION
[0043] As used herein, the term "chromatin modification domain"
(CMD) refers to nucleotide sequences that interact with a variety
of proteins associated with maintaining and/or altering chromatin
structure.
[0044] As used herein, the term "cloning" refers to the process of
ligating a DNA molecule into a plasmid and transferring it an
appropriate host cell for duplication during propagation of the
host.
[0045] As used herein, the terms "cloning vector" and "cloning
vector plasmid" are used interchangeably to refer to a circular DNA
molecule minimally containing an Origin of Replication, a means for
positive selection of host cells harboring the plasmid such as an
antibiotic-resistance gene; and a multiple cloning site.
[0046] As used herein, the term "common" in relation to
endonuclease sites refers to any endonuclease site that occurs
relatively frequently within a genome.
[0047] As used herein, the phrase "compatible to" refers a terminus
or end, either 5' or 3', of a strand of DNA which can form hydrogen
bonds with any other complementary termini either cleaved with the
same restriction enzyme or created by some other method. Since any
DNA that contains a specific recognition sequence for a restriction
enzyme will be cut in the same manner as any other DNA containing
the same sequence, those cleaved ends will be complementary and
thus compatible. Therefore, the ends of any DNA molecules cut with
the same restriction enzyme "match" each other in the way adjacent
pieces of a jigsaw puzzle "match", and can be enzymatically linked
together. Compatible ends will form a recognition site for a
particular restriction enzyme when combined together.
[0048] As used herein, the term "de novo synthesis" refers to the
process of synthesizing double-stranded DNA molecules of any length
by linking complementary single-stranded DNA molecules compatible
overhangs that represent subsequences of the total desired DNA
molecule.
[0049] As used herein, the term "DNA construct" refers to a DNA
molecule synthesized by consecutive cloning steps within a cloning
vector plasmid, and is commonly used to direct gene expression in
any appropriate cell host such as cultured cells in vitro, or a
transgenic mouse in vivo. A transgene used to make such a mouse can
also be referred to as a DNA construct, especially during the
period of time when the transgene is being designed and
synthesized.
[0050] As used herein, the term "DNA fragment" refers to any
isolated molecule of DNA, including but not limited to a
protein-coding sequence, reporter gene, promoter, enhancer, intron,
exon, poly-A tail, multiple cloning site, nuclear localization
signal, or mRNA stabilization signal, or any other naturally
occurring or synthetic DNA molecule, or any portion thereof.
Alternatively, a DNA fragment may be completely of synthetic
origin, produced in vitro. Furthermore, a DNA fragment may comprise
any combination of isolated naturally occurring and/or synthetic
fragments.
[0051] As used herein, the term "Docking Plasmid" refers to a
specialized cloning vector plasmid used in the invention to
assemble DNA fragments into a DNA construct.
[0052] As used herein, the terms "endonuclease" or "endonuclease
enzyme" refers to a member or members of a classification of
catalytic molecules that bind a recognition site encoded in a DNA
molecule and cleave the DNA molecule at a precise location within
or near the sequence.
[0053] As used herein, the terms "endonuclease recognition site",
recognition site", "cognate sequence" or "cognate sequences" refer
to the minimal string of nucleotides required for a restriction
enzyme to bind and cleave a DNA molecule or gene.
[0054] As used herein, the term "enhancer region" refers to a
nucleotide sequence that is not required for expression of a target
gene, but will increase the level of gene expression under
appropriate conditions.
[0055] As used herein, the term "gene expression host selector
gene" (GEH-S) refers to a genetic element that can confer to a host
organism a trait that can be selected, tracked, or detected by
optical sensors, PCR amplification, biochemical assays, or by
cell/organism survival assays (resistance or toxicity to cells or
organisms when treated with an appropriate antibiotic or
chemical).
[0056] As used herein, the terms "gene promoter" or "promoter"
refer to a nucleotide sequence required for expression of a gene,
or any portion of the full-length promoter.
[0057] As used herein, the terms "insert" and "module" are
essentially interchangeable, with the only fine distinction being
that an "insert" is inserted into the vector, and once it is
inserted it is then more commonly called a "module". A module can
then be removed from the vector. Also, the term insert is commonly
used for an isolated module used as an insert into a modular
acceptor vector.
[0058] As used herein, the term "intron" refers to the nucleotide
sequences of a non-protein-coding region within a mammalian cell
gene found between two protein-coding regions or exons.
[0059] As used herein, the term "localization signal" (LOC) refers
to nucleotide sequences encoding a signal for subcellular routing
of a protein of interest.
[0060] As used herein, the term "multiple cloning site" (MCS)
refers to nucleotide sequences comprising at least one unique
endonuclease site, and, more typically, a grouping of unique
endonuclease sites, for the purpose of cloning DNA fragments into a
cloning vector plasmid
[0061] As used herein, the term "mRNA stabilization element" refers
a sequence of DNA that is recognized by binding proteins thought to
protect some mRNAs from degradation.
[0062] As used herein, the term "Origin of Replication" (ORI)
refers to nucleotide sequences that direct replication or
duplication of a plasmid within a host cell.
[0063] As used herein, the phrase "PCR terminator over-hang cloning
technology" refers to the process of amplifying genetic modules
using the polymerase chain reaction in conjunction with
single-stranded DNA primers with protected 5'over-hanging
nucleotides that can serve as junction sites with complementary DNA
over-hangs.
[0064] As used herein, the term "poly-A tail" refers to a sequence
of adenine (A) nucleotides commonly found at the end of messenger
RNA (mRNA) molecules. A Poly-A tail signal is incorporated into the
3' ends of DNA constructs or transgenes to facilitate expression of
the gene of interest.
[0065] As used herein, the term "primer site" refers to nucleotide
sequences that serve as DNA templates onto which single-stranded
DNA oligonucleotides can anneal for the purpose of initiating DNA
sequencing, PCR amplification, and/or RNA transcription.
[0066] As used herein, the term "pUC19" refers to a plasmid cloning
vector well-known to those skilled in the art, and can be found in
the NCBI Genbank database as Accession # L09137.
[0067] As used herein, the term "random nucleotide sequences"
refers to any combination of nucleotide sequences that do not
duplicate sequences encoding other elements specified as components
of the same molecule. The number of nucleotides required in the
random sequences is dependent upon the requirements of the
endonuclease enzymes that flank the random sequences. Most
endonucleases require a minimum of 2-4 additional random sequences
to stabilize DNA binding. It is preferred that the number of random
sequences would be a multiple of 3, corresponding to the number of
nucleotides that make up a codon. The preferred minimum number of
random sequences is therefore 6, however, fewer or more nucleotides
may be used.
[0068] As used herein, the term "rare" in relation to endonuclease
sites refers to an endonuclease site that occurs relatively
infrequently within a genome.
[0069] As used herein, the term "recombination arm" refers to
nucleotide sequences that facilitate the homologous recombination
between transgenic DNA and genomic DNA. Successful recombination
requires the presence of a left recombination arm (LRA) and a right
recombination arm (RRA) flanking a region of transgenic DNA to be
incorporated into a host genome via homologous recombination.
[0070] As used herein, the term "recombineering" refers to the
process of using random or site-selective recombinase enzymes in
conjunction with DNA sequences that can be acted on by recombinase
enzymes to translocate a portion of genetic material from one DNA
molecule to a different DNA molecule.
[0071] As used herein, the term "reporter gene" refers to a
nucleotide sequences encoding a protein useful for monitoring the
activity of a particular promoter of interest.
[0072] As used herein, the term "Shuttle Vector" refers to a
specialized cloning vector plasmid used in the invention to make an
intermediate molecule that will modify the ends of a DNA
fragment.
[0073] As used herein, the term "tag sequence" (TAG) refers to
nucleotide sequences encoding a unique protein region that allows
it to be detected, or in some cases, distinguished from any
endogenous counterpart.
[0074] As used herein, the term "untranslated region" (UTR) refers
to nucleotide sequences encompassing the non-protein-coding region
of an mRNA molecule. These untranslated regions can reside at the
5' end (5' UTR) or the 3' end (3' UTR) an mRNA molecule.
[0075] The present invention provides a method to take a newly
manufactured transgene containing the modules and selectively
remove one or more of the module and replace it with a different
insert. This process is called herein "second pass" and "multiple
threading". The invention further provides a method for creating an
array of different transgenes, each having a different Promoter,
Expression and Regulatory insert, by incorporating multiple
different Promoter, Expression and Regulatory inserts into a
cloning vector plasmid in a single step. The present invention also
provides a method that comprises the steps of providing cloning
vector plasmids having newly introduced Promoter, Expression and
Regulatory inserts combined together, removing the entire
combination as a backbone vector, and inserting a multiple number
of backbone vectors into a single cloning vector plasmid.
[0076] The present invention also provides a method to create a
modular cloning vector plasmid for the synthesis of a transgene or
other complicated DNA construct by providing a backbone having
docking points therein. Each docking point represents an area in
which there is preferably at least one fixed non-variable rare
endonuclease site, and more preferably fixed groupings of two
non-variable rare endonuclease sites, and most preferably fixed
groupings of three non-variable rare endonuclease sites. A
particular restriction site of each docking point is cleaved by its
cognate endonuclease enzyme. This will create either a desired 5'
or 3' end which is compatible with the complementary 5' or 3' end
of one of the pre-constructed inserts containing a nucleotide
sequence of choice, such as a Promoter, Expression or Regulator
nucleotide sequence. At least two inserts, each of which have 5'
and 3' ends that are compatible with the cleaved docking point of
interest, can be added along with the cleaved cloning vector
plasmid to an appropriate reaction mixture, and, assuming the
proper thermodynamic milieu, the inserts can simultaneously, i.e.
in a single step, become integrated into the cloning vector
plasmid. During this singular addition and ligation reaction, the
docking points are reformed and the cloning vector plasmid becomes
modular, in that the docking points and the connection between the
two modules can be re-cleaved with the appropriate restriction
enzymes. The module can then later be removed, and a new module can
be put in its place.
[0077] One embodiment of the present invention relates to a method
for constructing a transgene, comprising the steps of providing a
cloning vector plasmid with a backbone able to accept a sequential
arrangement of inserts, providing at least a first insert and a
second insert to be included in the transgene, and transferring
both the first insert and the second insert to the backbone in a
single reaction. More preferably the inserts consist of three
inserts, specifically at least one Promoter, Expression, and
Regulatory module.
[0078] Another embodiment of the invention is a method for making a
transgene comprising the steps of providing a cloning vector
plasmid comprising first and second docking points, introducing
Promoter nucleotide sequences to be included in the transgene into
a Promoter shuttle vector, introducing Expression nucleotide
sequences to be included in the transgene into an Expression
shuttle vector, introducing Regulatory nucleotide sequences to be
included in the transgene into a Regulatory shuttle vector,
transferring simultaneously the Promoter, Expression and Regulatory
nucleotide sequences from the Promoter, Expression and Regulatory
shuttle vectors to the cloning vector plasmid, between the first
and second docking points.
[0079] It is preferred that both the 5' and 3' ends of each of the
docking points and each of the inserts all are compatible with a
corresponding end of another docking point or insert. For example,
if a first docking point contains a restriction site for a
non-variable rare restriction enzyme such as SgrAI and that docking
point is thereafter cleaved, then a first insert intended to be
inserted at the 3' end of the cleaved first docking point will
contain a compatible 5' end to create a restriction site for SgrAI
when the insert is combined with the first docking point. A second
docking point within the plasmid may, for example, have a
restriction site for a non-variable restriction enzyme such as
SwaI. Any second insert will have at its 3' end a compatible
nucleotide sequence to combine with the cleaved 5' end of the
cleaved second docking point to create a restriction site for SwaI.
Further, the 3' end of the first insert and the 5' end of the
second insert, in order to simultaneously be inserted into the
modular cloning vector plasmid and also thereafter be removed at
the same point, must contain compatible ends to create a third
restriction site for a third non-variable rare restriction enzyme,
such as PacI or SalI.
[0080] Sequential elements encoding the modular structure of the
present invention can specifically comprise: three non-variable and
unique common restriction sites, a 5' oligonucleotide primer site,
a unique HE site in a forward orientation, a pair of non-variable
and unique, common restriction sites flanking random nucleotide
sequences, a fixed grouping of non-variable rare restriction sites
to define a 5' portion of a promoter module, random nucleotide
sequences, a fixed grouping of non-variable rare restriction sites
that define a shared junction between a 3' position relative to the
Promoter/intron module and a 5' position relative to an Expression
module, random nucleotide sequences, a fixed grouping of
non-variable rare restriction sites that define a junction of a 3'
position relative to the Expression module and a 5' position
relative to a 3' Regulatory module, random nucleotide sequences, a
fixed grouping of non-variable rare restriction sites that define a
3' position relative to a 3' Regulatory module, a pair of
non-variable and unique, common restriction sites flanking random
nucleotide sequences, a unique HE site in reverse orientation that
is the same HE site as that placed 3' of the 5' oligonucleotide
primer site, a 3' oligonucleotide primer site in reverse
orientation, and four non-variable and unique common restriction
sites that define a 3' insertion site.
[0081] Other sequential elements encoding the modular structure of
the present invention can specifically comprise: two non-variable
and unique common restriction sites that define a 5' insertion
site, an oligonucleotide primer site, a pair of unique HE sites in
opposite orientation flanking random nucleotide sequences, a
non-variable and unique, common restriction site that allows
cloning of a shuttle vector module downstream of the pair of unique
HE sites, a fixed grouping of non-variable rare restriction sites,
random nucleotide sequences, a fixed grouping of non-variable rare
restriction sites, a unique HE site in a forward orientation, a
pair of non-variable and unique, common restriction sites flanking
random nucleotide sequences, an oligonucleotide primer site, a pair
of unique BstX I sites in opposite orientations (wherein the
variable nucleotide region in the BstX I recognition site is
defined by nucleotides identical to the non-complementary tails
generated by the ordering of two identical HE recognition sites
arranged in reverse-complement orientation, a pair of unique HE
sites in opposite orientations flanking random nucleotide
sequences; an oligonucleotide primer site in reverse-orientation, a
pair of non-variable and unique, common restriction sites flanking
random nucleotide sequences, a unique HE site in reverse
orientation, with the HE site being the same as the HE site in a
forward orientation, a fixed grouping of non-variable rare
restriction sites, random nucleotide sequences, a fixed grouping of
non-variable rare restriction sites, a non-variable and unique,
common restriction site, a pair of unique HE sites in opposite
orientation flanking random nucleotide sequences, an
oligonucleotide primer site in reverse orientation, and three
non-variable and unique common restriction sites.
[0082] The present invention is a group of methods for assembling a
variety of DNA fragments into a de novo DNA construct or transgene
by using cloning vectors optimized to reduce the amount of
manipulation frequently needed.
[0083] The primary vector, herein referred to as a Docking Plasmid,
contains a multiple cloning site (MCS) with preferably 3 sets of
rare endonuclease sites arranged in a linear pattern. This
arrangement defines a modular architecture that allows the user to
assemble multiple inserts into a single transgene construct without
disturbing the integrity of DNA elements already incorporated into
the Docking Plasmid in previous cloning steps.
[0084] Two recognition sites for at least three HE are placed in
opposite orientation to flank three modular regions for the purpose
of creating a gene cassette acceptor site that cannot self-anneal.
Because HE sites are asymmetric and non-palindromic, it is possible
to generate non-complementary protruding 3' cohesive tails by
placing two HE recognition sites in opposite orientation. Thus, the
HEI-SceI cuts its cognate recognition site as indicated by "/":
TABLE-US-00004 5'...T A G G G A T A A / C A G G G T A A T...3',
3'...A T C C C / T A T T G T C C C A T T A...5'.
[0085] The reverse placement of a second site within an MCS would
generate two non-complementary cohesive protruding tails:
TABLE-US-00005 5'...TAGGGATAA CCCTA...3'
3'...ATCCCAATAGGGAT...5'.
[0086] This is particularly useful when it is necessary to subclone
larger transgenes into a vector. Due to the size of the insert, it
is thermodynamically more favorable for a vector to self anneal
rather than accept a large insert. The presence of
non-complementary tails generated by this placement of restriction
sites provides chemical forces to counteract the thermodynamic
inclination for self-ligation.
[0087] The asymmetric nature of most HE protruding tails also
creates a powerful cloning tool when used in combination with the
BstX I endonuclease enzyme site (5'CCANNNNN/NTGG 3', where `N` can
be any nucleotide). The sequence-neutral domain of BstX I can be
used to generate compatible cohesive ends for two reverse-oriented
HE protruding tails, while precluding self-annealing.
[0088] BstX I (I-Sce I Fwd.) I-Sce I Forward I-Sce I Reverse BstX I
(I-Sce I Rev.)
TABLE-US-00006 5'-CCAGATAA CAGGGTAAT//ATTACCCTGTTAT GTGG-3' 3'-GGTC
TATTGTCCCATTA//TAATGGGAC AATACACC-5'
[0089] Endonuclease sites used in the invention were chosen
according to a hierarchy of occurrence. In order to determine the
frequency of endonuclease site occurrence, DNA sequence information
corresponding to nineteen different genes was analyzed using Vector
NTI software. This search covered a total of 110,530 nucleotides of
DNA sequence. Results from these analyses were calculated according
to the number of instances of an endonuclease site occurring within
the analyzed 110,530 nucleotides. Endonuclease sites were then
assigned a hierarchical designation according to four
classifications, wherein "common" sites occur greater than 25 times
per 110,530 nucleotides, "lower-frequency 6 bp sites" occur between
6-24 times per 110,530 nucleotides, and "rare" sites occur between
0-5 times per 110,530 nucleotides. A partial list of "suitable"
enzymes is hereby listed according to their occurrence
classifications:
[0090] Common endonuclease enzymes:
Ase I, BamH I, Bgl II, Bip I, BstX I, EcoR I, Hinc II, Hind III,
Nco I, Pst I, Sac I, Sac II, Sph I, Stu I, Xba I
[0091] Endonuclease enzymes that have a 6 bp recognition site, but
have a lower frequency of occurrence:
Aar I, Aat II, Afl II, Age I, ApaL I, Avr II, BseA I, BspD I, BspE
I, BstB I, Cla I, Eag I, Eco0109 I, EcoR V, Hpa I, Kpn I, Mfe I,
Nar I, Nde I, NgoM IV, Nhe I, Nsi I, Pml I, SexA, Sma I, Spe I, Xho
I
[0092] Rare endonuclease enzymes:
Acl I, Asc I, AsiS I, BsiW I, Fse I, Mlu I, Not I, Nru I, Pac I,
Pme I, Pvu I, Rsr II, Sal I, Sbf I, Sfi I, SgrA I, SnaB I, Swa I,
PI-Sce I, I-Sce I, I-Ceu I, PI-Psp I, I-Ppo I, I-Tli I
[0093] Other endonucleases not included in these listings can also
be used, maintaining the same functionality and the spirit and
intent of the invention.
[0094] The secondary vectors of the invention, herein known as
Shuttle vectors, contain multiple cloning sites with common
endonuclease sites flanked by rare endonuclease sites. The shuttle
vectors are designed for cloning fragments of DNA into the common
endonuclease sites between the rare sites. The cloned fragments can
subsequently be released by cleavage at the rare endonuclease site
or sites, and incorporated into the Docking Plasmid using the same
rare endonuclease site or sites found in the shuttle vectors.
[0095] Thus, unlike conventional cloning vectors, the design of the
MCS allows "cassettes" or modules of DNA fragments to be inserted
into the modular regions of the Docking Plasmid. Likewise, each can
be easily removed using the same rare endonuclease enzymes, and
replaced with any other DNA fragment of interest. This feature
allows the user to change the direction of an experimental project
quickly and easily without having to rebuild the entire DNA
construct. Thus, the cloning vector plasmids of the present
invention allow the user to clone a DNA fragment into an
intermediate vector using common endonuclease sites, creating a
cassette-accepting module, and to then transfer that fragment to
the desired modular spot in the final construct by means of rare
endonuclease sites. Furthermore, it allows future alterations to
the molecule to replace individual modules in the Docking Plasmid
with other cassette modules. The following descriptions highlight
distinctions of the present invention compared with the prior
art.
[0096] Individual components of a transgene (the promoter enhancer
P, expressed protein E, and/or 3' regulatory region 3) can be
assembled as modules transferred from shuttle vectors into the PE3
Docking Station Plasmid. If higher orders of complexity are needed,
the assembled transgenes, or other nucleotide sequences, can then
be transferred into a Primary Docking Plasmid. Each of the five
types of cloning vector plasmids will be explained in greater
detail to provide an understanding of the components incorporated
into each, beginning with the more complex PE3 Docking Station
Plasmid and the Primary Docking Plasmid.
[0097] The PE3 Docking Plasmid comprises a pUC19 backbone with the
following modifications, wherein the sequences are numbered
according to the pUC19 Genbank sequence file, Accession #
L09137:
[0098] 1. Only sequences from 806 to 2617 (Afl3-Aat2) are used in
the Docking Plasmid,
[0099] 2. The BspH1 site at 1729 in pUC19 is mutated from TCATGA to
GCATGA,
[0100] 3. The Acl1 site at 1493 in pUC19 is mutated from AACGTT to
AACGCT,
[0101] 4. The Acl1 site at 1120 in pUC19 is mutated from AACGTT to
CACGCT,
[0102] 5. The Ahd1 site in pUC19 is mutated from GACNNNNNGTC to
CACNNNNNGTC,
[0103] 6. Sequences encoding BspH1/I-Ppo 1/BspH1 are inserted at
the only remaining BspH1 site in pUC19 following the mutation step
2 in the list above.
[0104] The multiple cloning site (MCS) in the PE3 Docking Plasmid
comprises the following sequential elements, in the order
listed:
[0105] 1. Three non-variable and unique common endonuclease sites
that define a 5' insertion site for the mutated pUC19 vector
described above (shown as, but not limited to, Aat II, Blp I, and
EcoO109 I);
[0106] 2. A T7 primer site;
[0107] 3. A unique HE site (for example, I-SceI (forward
orientation));
[0108] 4. A pair of non-variable and unique, common endonuclease
sites flanking random nucleotide sequences that can serve as a
chromatin modification domain acceptor module (RNAS-CMD-1) (for
example, Kpn I and Avr II);
[0109] 5. A fixed grouping of non-variable rare endonuclease sites
that define the 5' portion of the promoter module (for example,
AsiS I, Pac I, and Sbf I);
[0110] 6. Random nucleotide sequences that can serve as a
Promoter/intron acceptor module (RNAS-P);
[0111] 7. A fixed grouping of non-variable rare endonuclease sites
that define the shared junction between the 3' portion of the
Promoter/intron module and the 5' portion of the Expression module
(for example, SgrA I, AscI, and MIuI);
[0112] 8. Random nucleotide sequences that can serve as an
expression acceptor module (RNAS-E);
[0113] 9. A fixed grouping of non-variable rare endonuclease sites
that define the junction of the 3' portion of the Expression module
and the 5' portion of the 3' Regulatory module (for example, SnaB
I, Not I, and Sal I);
[0114] 10. Random nucleotide sequences that can serve as a 3'
regulatory domain acceptor module (RNAS-3);
[0115] 11. A fixed grouping of non-variable rare endonuclease sites
that define the 3' portion of the 3' Regulatory module (for
example, Swa I, Rsr II, and BsiW I);
[0116] 12. A pair of non-variable and unique, common endonuclease
sites flanking a random nucleotide sequence of DNA that can serve
as a chromatin modification domain acceptor module (RNAS-CMD-2)
(for example, Xho I and Nhe I);
[0117] 13. A unique HE site in reverse orientation that is
identical to that in item 3, above;
[0118] 14. A T3 primer site in reverse orientation; and
[0119] 15. Four non-variable and unique common endonuclease sites
that define a 3' insertion site for the mutated pUC19 vector
described above (for example, BspE I, Pme I, Sap I, and BspH
I).
[0120] The Primary Docking Plasmid can be used to assemble two
completed transgenes that are first constructed in PE3 Docking
Station Plasmids, or two homology arms needed to construct a
gene-targeting transgene, or to introduce two types of positive or
negative selection elements. The multiple cloning site (MCS) in the
Primary Docking Plasmid comprises the following sequential
elements, in the order listed:
[0121] 1. Two non-variable and unique common endonuclease sites
that define a 5' insertion site for the mutated pUC19 vector
described above (for example, Aat II and Blp I);
[0122] 2. An M13 Rev. primer site;
[0123] 3. A pair of unique endonuclease flanking a random
nucleotide sequence of DNA that can serve as a genome expression
host selector gene acceptor module (RNAS-GEH-S1);
[0124] 4. A non-variable and unique, common endonuclease site that
allows cloning of a shuttle vector module downstream of the HE pair
(for example, EcoO109I);
[0125] 5. A fixed grouping of non-variable rare endonuclease sites
that define the 5' portion a Left Recombination Arm module (for
example, AsiS I, Pac I, and Sbf I);
[0126] 6. Random nucleotide sequences that can serve as a Left
Recombination Arm acceptor module (RNAS-LRA);
[0127] 7. A fixed grouping of non-variable rare endonuclease sites
that define the 3' portion of the Left Recombination Arm acceptor
module (for example, SgrA I, MIuI, and AscI);
[0128] 8. A unique HE site (for example, I-Ceu I (forward
orientation));
[0129] 9. A pair of non-variable and unique, common endonuclease
sites flanking a random nucleotide sequence of DNA that can serve
as a chromatin modification domain acceptor module (RNAS-CMD-1)
(for example, Kpn I and Avr II);
[0130] 10. A T7 primer site;
[0131] 11. A pair of unique BstX I sites in opposite orientation
(wherein the variable nucleotide region in the BstX I recognition
site is defined by nucleotides identical to the non-complementary
tails generated by the ordering of two identical HE recognition
sites arranged in reverse-complement orientation; for example,
PI-SceI (forward orientation) and PI-SceI (reverse orientation))
flanking a random nucleotide sequence of DNA that can serve as a
complex transgene acceptor module (RNAS-PE3-1);
[0132] 12. A pair of unique endonuclease sites flanking a random
nucleotide sequence of DNA that can serve as a complex transgene
acceptor module (RNAS-PE3-2);
[0133] 13. A T3 primer site in reverse-orientation;
[0134] 14. A pair of non-variable and unique, common endonuclease
sites flanking a random nucleotide sequence of DNA that can serve
as a chromatin modification domain acceptor module (RNAS-CMD-2)
(for example, Xho I and Nhe I);
[0135] 15. A unique HE site in reverse orientation that is
identical to that in item 8 above;
[0136] 16. A fixed grouping of non-variable rare endonuclease sites
that define the 5' portion a Right Recombination Arm module (for
example, SnaB I, Sal I, and Not I);
[0137] 17. Random nucleotide sequences that can serve as a Right
Recombination Arm acceptor module (RNAS-RRA);
[0138] 18. A fixed grouping of non-variable rare endonuclease sites
that define the 3' portion of the Right Recombination Arm acceptor
module (for example, Rsr II, Swa I, and BsiW I);
[0139] 19. A non-variable and unique, common endonuclease site that
allows cloning of a shuttle vector module (for example, BspE
I);
[0140] 20. A pair of unique endonuclease sites flanking a random
nucleotide sequence of DNA that can serve as a genome expression
host selector gene acceptor module (RNAS-GEH-S2);
[0141] 21. An M13 Forward primer site placed in reverse
orientation; and
[0142] 22. Three non-variable and unique common endonuclease sites
that define a 3' insertion site for the mutated pUC19 vector
described above (for example, Pme I, Sap I, and BspH I).
[0143] Three cloning vector plasmids of the invention are known as
Shuttle Vectors. The Shuttle Vectors, like the PE3 and Primary
Docking Plasmids, are also constructed from a pUC19 backbone. Just
like the PE3 and Primary Docking Plasmids, each Shuttle Vector has
the same modifications to the pUC19 backbone listed as 1 through 6,
above. The individual Shuttle Vectors (SV) are identified as
Shuttle Vector Promoter/intron (P), Shuttle Vector Expression (E),
and Shuttle Vector 3'Regulatory (3); henceforth SVP, SVE, and SV3,
respectively. Each is described more fully below.
[0144] Shuttle Vector P(SVP):
[0145] SVP is a cloning vector plasmid that can be used to prepare
promoter and intron sequences for assembly into a transgene
construct. An example of an SVP Plasmid can comprise the following
sequential elements in the MCS, in the order listed:
[0146] 1. Two non-variable and unique, common endonuclease sites
that define a 5' insertion site for the mutated pUC19 vector
described above (for example, AatII and BlpI);
[0147] 2. A T7 primer site;
[0148] 3. A non-variable and unique, common endonuclease site that
allows efficient cloning of a shuttle vector module downstream of
the T7 primer site (for example, Eco0109I);
[0149] 4. A fixed grouping of non-variable rare endonuclease sites
that define the 5' portion of the promoter module (for example,
AsiSI, Pac I, and Sbf I). These non-variable rare endonuclease
sites provide the docking point represented by the star at the 5'
end of the Promoter Vector of FIG. 2;
[0150] 5. A variable MCS comprising any grouping of common or rare
endonuclease sites that are unique to the shuttle vector;
[0151] 6. A fixed grouping of non-variable rare endonuclease sites
that define the 3' portion of the promoter module (for example,
SgrA I, AscI, and MiuI). These non-variable rare endonuclease sites
provide the docking point represented by the circle at the 3' end
of the Promoter Vector of FIG. 2;
[0152] 7. A non-variable and unique, common endonuclease site that
allows efficient cloning of a shuttle vector module upstream of the
T3 primer site (for example, BspEI);
[0153] 8. A reverse-orientation T3 primer site; and
[0154] 9. Two non-variable and unique, common endonuclease sites
that define a 3' insertion site for the mutated pUC19 vector
described above (for example, PmeI and SapI).
[0155] Shuttle Vector E (SVE):
[0156] This is a cloning vector plasmid that can be used to prepare
sequences to be expressed by the transgene for assembly into a
transgene construct. An example of an SVE plasmid can comprise the
following sequential elements in the MCS, in the order listed:
[0157] 1. Two non-variable and unique, common endonuclease sites
that define a 5' insertion site for the mutated pUC19 vector
described above (for example, AatII and Blp I);
[0158] 2. A T7 primer site;
[0159] 3. A non-variable and unique, common endonuclease site that
allows efficient cloning of a shuttle vector module downstream of
the T7 primer site (for example, Eco0109I);
[0160] 4. A fixed grouping of non-variable rare endonuclease sites
that define the 5' portion of the expression module (for example,
SgrA I, AscI, and MIuI). These non-variable rare endonuclease sites
provide the docking point represented by the circle at the 5' end
of the Expression Vector of FIG. 2;
[0161] 5. A variable MCS consisting of any grouping of common or
rare endonuclease sites that are unique to the shuttle vector;
[0162] 6. A fixed grouping of non-variable rare endonuclease sites
that define the 3' portion of the expression module (for example,
SnaBI, NotI, and SalI). These non-variable rare endonuclease sites
provide the docking point represented by the triangle at the 3' end
of the Expression Vector of FIG. 2;
[0163] 7. A non-variable and unique, common endonuclease site that
allows efficient cloning of a shuttle vector module upstream of the
T3 primer site (for example, BspEI);
[0164] 8. A reverse-orientation T3 primer site; and
[0165] 9. Two non-variable and unique, common restriction sites
that define a 3' insertion site for the mutated pUC19 vector
described above (for example, PmeI and SapI).
[0166] Shuttle Vector 3 (SV3):
[0167] This is a cloning vector plasmid that can be used to prepare
3' regulatory sequences for assembly into a transgene construct. An
example of an SV3 plasmid can comprise the following elements in
the MCS, in the order listed:
[0168] 1. Two non-variable and unique, common endonuclease sites
that define a 5' insertion site for the mutated pUC19 vector
described above (for example, AatII and BlpI);
[0169] 2. A T7 primer site;
[0170] 3. A non-variable and unique, common endonuclease site that
allows efficient cloning of a shuttle vector module downstream of
the T7 primer (for example, Eco0109I);
[0171] 4. A fixed grouping of non-variable rare endonuclease sites
that define the 5' portion of the 3' regulatory module (for
example, SnaBI, NotI, and SalI). These non-variable rare
endonuclease sites provide the docking point represented by the
triangle at the 5' end of the Regulatory Vector of FIG. 2.
[0172] 5. A variable MCS consisting of any grouping of common or
rare endonuclease sites that are unique to the shuttle vector;
[0173] 6. A fixed grouping of non-variable rare endonuclease sites
that define the 3' portion of the 3' regulatory module (for
example, SwaI, RsrII, and BsiWI). These non-variable rare
endonuclease sites provide the docking point represented by the
square at the 3' end of the Regulatory Vector of FIG. 2;
[0174] 7. A non-variable and unique, non-rare endonuclease site
that allows efficient cloning of a shuttle vector module upstream
of the T3 primer site (for example, BspEI);
[0175] 8. A reverse-orientation T3 primer site; and
[0176] 9. Two non-variable and unique, non-rare endonuclease sites
that define a 3' insertion site for the mutated pUC19 vector
described above (for example, PmeI and SapI).
[0177] While the present invention discloses methods for building
transgenes in plasmid cloning vectors, similar methods can be used
to build transgenes in larger extrachromosomal DNA molecules such
as cosmids or artificial chromosomes, including bacterial
artificial chromosomes (BAC). For use in plants, a T1 vector may
also be used. The wide variety of genetic elements that can be
incorporated into the plasmid cloning vectors also allow transfer
of the final transgene products into a wide variety of host
organisms with little or no further manipulation.
[0178] FIGS. 2 and 3 are a general illustration of the modularity
of the invention. As shown in FIG. 2, there is one each of a
Promoter, Expression, and 3' Regulatory shuttle vector. Flanking
each insert within the shuttle vectors are endonuclease restriction
sites that are specific for creating a docking point. More
specifically, in FIG. 2, the Promoter insert (P) is flanked by a
first group of one or more endonuclease restriction sites
represented by astar at the 5' end and a second group of one or
more endonuclease restriction sites represented by a circle at the
3' end; the Expression module is flanked by the second group of
endonuclease restriction sites represented by the circle at the 5'
end and a third group of one or more endonuclease restriction sites
represented by a triangleat the 3' end; and the 3' Regulatory
module (3) is flanked by the third group of endonuclease
restriction sites represented by the triangle at the 5' end and a
fourth group of one or more endonuclease restriction sites
represented by a square at the 3' end. Cleaving each endonuclease
recognition site by the endonuclease specific for that site creates
sticky ends at the 5' and 3' end of each module, as indicated in
bottom portion of FIG. 2 by the inserts at the end of the dashed
line arrows. The modules can now be combined with a cloning vector
plasmid which has also been cleaved at its two fixed docking points
by endonucleases specific for the first group of endonuclease
restriction sites (represented by the star) and the fourth group of
endonuclease restriction sites (represented by the square). When
the modular vectors are placed with the cloning vector plasmid in
an appropriate reaction mixture, the cleaved sticky ends
(represented by hollow stars, circles, triangles and squares) of
each modular vector will self-orient within the plasmid and
sequentially ligate, with the cleaved star ends combining, the
cleaved circle ends combining, the cleaved triangle ends combining,
and the cleaved square ends combining. This results in an assembled
backbone vector shown in FIG. 3. Further, each of the combined
groups of endonuclease sites represented by the star, circle,
triangle, and square can once again be cleaved by its corresponding
specific endonuclease, such that a particular insert can later be
removed and replaced with another insert of interest.
[0179] Multiple backbone vectors (example, PE3-1 and PE3-2) can be
inserted into a single docking plasmid. The asymmetric nature of
the protruding tails of an endonuclease such as I-Sce I, as with
other HE's, creates a powerful cloning tool when used in
combination with the BstX I endonuclease enzyme site (5'
CCANNNNN/NTGG 3', where `N` can be any nucleotide). The
sequence-neutral domain of BstX I can be used to generate
compatible cohesive ends for two reverse-oriented I-Sce I
protruding tails, while precluding self-annealing. With this
method, a first insert, PE3-1, having an I-Sce-1 site at its ends
can be placed in a cloning vector plasmid by cleaving the plasmid
at the Bstx1/Sce1 endonuclease sites. One can then cut again with
I-SceI and insert a PE3-2 having I-Sce-1 site at its ends. This
entire backbone can then be cleaved from its docking plasmid by
PI-Sce I and inserted into another docking plasmid that contains
BstX I/PI-Sce I endonuclease sites. This second docking plasmid
also has endonuclease sites for PI-Sce I, into which yet another
module for a separate docking plasmid, possible containing a PE3-3
and PE3-4, can be inserted (not shown). In this manner, a
researcher can get more information into one cell, that is, one can
insert multiple genes within the context of a single vector, which
has not previously been accomplished by those skilled in the art.
Such a novel process can save a both money and time for researchers
working in this field.
EXAMPLES
Example 1
PE3 Docking Plasmid
[0180] As an example of the method of practicing the present
invention, a transgene can be constructed containing these
elements:
[0181] 1. Nucleotide sequences of the human promoter for surfactant
protein C(SP-C);
[0182] 2. Sequences encoding the protein product of the mouse gene
granulocyte-macrophage colony-stimulating factor-receptor beta c
(GMR.beta.c);
[0183] 3. Rabbit betaglobin intron sequences; and
[0184] 4. SV40 poly-A signal.
[0185] The SP-C sequences contain internal BamH1 sites, and can be
released from its parental plasmid only with Not1 and EcoR1.
GMR.beta.c has an internal Not1 site, and can be cut from its
parental plasmid with BamH1 and Xho1. The rabbit betaglobin intron
sequences can be cut out of its parental plasmid with EcoR1. The
SV-40 poly-A tail can be cut from its parental plasmid with Xho1
and Sac1. Because of redundancy of several of endonuclease sites,
none of the parental plasmids can be used to assemble all the
needed fragments.
[0186] The steps used to build the desired transgene in the PE3
Docking Plasmid invention are as follows.
[0187] 1. Since Not1 and PspOM1 generate compatible cohesive ends,
the human SP-C promoter sequences are excised with Not1 and EcoR1
and cloned into the PspOM1 and EcoR1 sites of Shuttle Vector P. The
product of this reaction is called pSVP-SPC
[0188] 2. Following propagation and recovery steps well known to
those skilled in the art, the rabbit betaglobin intron sequences
are cloned into the EcoR1 site of pSVP-SPC. Orientation of the
intron in the resultant intermediate construct is verified by
sequencing the product, called pSVP-SPC-r.beta.G.
[0189] 3. The promoter and intron are excised and isolated as one
contiguous fragment from pSVP-SPC-r.beta.G using AsiS1 and Asc1.
Concurrently, the PE3 Docking Plasmid is cut with AsiS1 and Asc1 in
preparation for ligation with the promoter/intron segment. The
promoter/intron fragment is ligated into the Docking Plasmid,
propagated, and recovered.
[0190] 4. The Xho1 site of the GMR.beta.c fragment is filled in to
create a blunt 3' end, using techniques well known to those skilled
in the art. It is then cloned into the BamH1 site and the
blunt-ended Pvu2 site of pSVP-SPC-r.beta.G. The resultant plasmid
(pDP-SPC-GMR.beta.-r.beta.G) was propagated and recovered.
[0191] 5. The final cloning step is the addition of the SV-40
Poly-A tail. The SV40-polyA fragment is cut out with Xho1 and Sac1,
as is the recipient vector pDS1-SPC-GMR.beta.c-rb.beta.G. Both
pieces of DNA are gel purified and recovered. A ligation mix is
prepared with a 10:1 molar ratio of SV-40polyA to
pDS1-SPC-GMR.beta.c-r.beta.G. The ligation products are propagated
and harvested. The new plasmid,
pDS1-SPC-GMR.beta..beta.c-r.beta.G-pA contains all elements
required for the transgene, including a unique endonuclease site at
the 3' end with which the entire pDS1-SPC-GMR.beta.c-r.beta.G-pA
plasmid can be linearized for transfection into eukaryotic cells or
microinjection into the pronucleus of a fertilized ovum.
Example 2
Dynamic Vector Assembly
[0192] Dynamic Vector Assembly is illustrated in the following
example:
[0193] 1. Promoter sequences from the human cytomegalovirus (CMV)
are inserted into a P Shuttle Vector (SVP), having AsiSI and Ase I
endonuclease at the 5' and 3' portions, respectively. Plasmids are
amplified, and the promoter module is cleaved from the vector by
AsiS I and Asc I endonuclease digestion and isolated.
[0194] 2. Sequences encoding a luciferase protein are inserted into
an Expression Shuttle Vector (SVE), having Asc I and Not I
endonuclease at the 5' and 3' portions, respectively. Plasmids are
amplified, and the Expression module is cleaved from the vector by
Asc I and Not I endonuclease digestion and isolated.
[0195] 3. Sequences encoding a mammalian intron and SV40
poly-adenylation site are inserted into a 3' Regulatory Shuttle
vector (SV3), having Not I and BsiW I endonuclease at the 5' and 3'
portions, respectively. Plasmids are amplified, and the Regulatory
module is cleaved from the vector by Not I and BsiW I endonuclease
digestion and isolated.
[0196] 4. The endonuclease recognition sites in a Docking Vector
plasmid having AsiS I and BsiW I endonuclease at the 5' portion of
the promoter module and the 3' portion of the regulator module,
respectively, are cleaved with AsiS I and BsiW I endonuclease and
isolated.
[0197] 5. The Promoter, Expression, and Regulatory modules are
combined with the Docking Vector Plasmid in a ligation mixture.
Following an incubation of 2 hours, the ligation mixture is used to
transform E. coli, which are then spread on an LB agar plate with
ampicillin. The plate is incubated at 37.degree. C. overnight.
Colonies are isolated and propagated in individual liquid LB broth
cultures. The plasmid DNA is isolated from each LB broth culture.
The DNA is analyzed by endonuclease mapping to determine whether
the plasmids from each colony contain the three modular inserts
(Promoter, Expression and Regulatory). A plasmid that contains the
three modular inserts is identified as the transgene pCMV-luc-SV40
pA. It can be linearized using I-Sce I endonuclease and injected
into mouse pronuclei to generate CMV-luciferase mice. The CMV
promoter in this example directs the expression of the luciferase
gene in all tissues of a host organism, such as a CMV-luciferase
mouse.
Example 3
Redesign of a Dynamic Vector Assembly
[0198] If the researcher now wishes to refine the expression
pattern so that luciferase is expressed only in a particular tissue
or cell-type, he or she can quickly and easily replace the CMV
promoter with one that will provide a restricted expression
pattern. The following example illustrates the use of the invention
to facilitate rapid redesign of pCMV-luc-pA:
[0199] 1. A neuron-specific promoter, Neuron-Specific Enolase
(NSE), is inserted into a P Shuttle Vector (SVP) and prepared as
the Promoter Module in the previous example.
[0200] 2. pCMV-luc-pA is cleaved with AsiS I and Asc I to remove
the CMV Promoter Module. The remainder of the Docking Vector
Plasmid containing intact Expression and Regulatory Modules is
isolated.
[0201] 3. The NSE Promoter Module is placed in a ligation mixture
with the remainder of the Docking Vector Plasmid containing intact
Expression and Regulatory Modules. Following incubation for 2
hours, the new ligation mixture is used to transform E. coli. The
E. coli mixture is spread on an LB agar plate with ampicillin, as
in the previous example. Colonies are isolated the following day,
propagated, and plasmid DNA is isolated from each. Endonuclease
mapping is used to identify plasmids that contain the desired NSE
Promoter module.
Example 4
Array of Transgenes
[0202] The following example is an illustration of the use of the
invention to rapidly assemble an array of transgenes, each
containing a different combination of Promoter, Expression, and
Regulatory modules. A series of six shuttle vectors and a PE3
docking station vector will be used to generate eight different
vector products using combinatorial assembly. The series of six
shuttle vectors consists of two P-Shuttles (SVP), two E-Shuttles
(SVE), and two 3-Shuttles (SV3). The two discrete P-Shuttles (SVP)
contain either a human cytomegalovirus (CMV) promoter or a mouse
SPC lung-specific promoter, and each has AsiS I and Asc I
endonuclease at the 5' and 3' portions, respectively. The two
discrete E-Shuttles contain either a Luciferase cDNA or an EGFP
cDNA, and each has Asc I and Not I endonuclease at the 5' and 3'
portions, respectively. The two discrete 3-Shuttle vectors contain
either an SV40 polyA signal or the 3' regulatory region of the
human growth hormone (hGH), and each has Not I and BsiW I
endonuclease at the 5' and 3' portions, respectively.
[0203] The promoter modules are released from their respective SVP
shuttle vectors by individually digesting appropriate shuttle
vector with the AsiS I and the Asc I endonucleases. The resulting
restriction products are individually subjected to gel
electrophoresis and the DNA band corresponding to the appropriate
promoter module is subjected to gel purification. This procedure
will yield either a CMV promoter module or an SPC promoter module
bounded on the 5' side by an AsiS I overhang and by an Asc I
overhang on the 3' end.
[0204] The expression modules are released from their respective
SVE shuttle vectors by individually digesting appropriate shuttle
vector with the Asc I and the Not I restriction endonucleases. The
resulting restriction products are individually subjected to gel
electrophoresis and the DNA band corresponding to the appropriate
expression module is subjected to gel purification. This procedure
will yield either a Luciferase expression module or an EGFP
expression module bounded on the 5' side by an Asc I overhang and
by a Not I overhang on the 3' end.
[0205] The 3' regulatory modules are released from their respective
SV3 shuttle vectors by individually digesting appropriate shuttle
vector with the Not I and the BsiW I restriction endonucleases. The
resulting restriction products are individually subjected to gel
electrophoresis and the DNA band corresponding to the appropriate
3' regulatory module is subjected to gel purification. This
procedure will yield either a SV40 3' regulatory module or an hGH
3' regulatory module bounded on the 5' side by a Not I overhang and
by a BsiW I overhang on the 3' end.
[0206] The PE3 docking station vector is prepared by digesting with
the AsiS I and the BsiW I restriction endonucleases. To help
prevent future vector re-ligation, the vector restriction digest is
exposed to calf intestinal phosphatase (CIP) for one hour at
37.degree. C. The resulting CIP-treated vector restriction product
is then subjected to gel electrophoresis and the DNA band
corresponding to linearized PE3 vector backbone is subjected to gel
purification.
[0207] Samples from the seven resulting gel-purified DNA fragments
are analyzed for identity, integrity, purity, and quantity by
running out on a diagnostic electrophoretic gel. Quantitative data
concerning the relative abundance of the purified PE3 docking
station vector and the respective DNA modules is used to define the
amount of each component needed for a combinatorial ligation
reaction.
[0208] When setting up a ligation reaction, there are two
strategies that frequently lead to successful results. The first
strategy is to set up ligation reaction mixtures wherein the
insert-to-vector ratio is about 3:1. The second strategy, used when
more than one insert is being introduced to a single vector
simultaneously, is to introduce a molar equivalent of each genetic
module that will be inserted into the vector. This can be achieved
either by adding a variable volume of the modules to a reaction
container in order to obtain molar equivalence in the context of
the ligation reaction mixture, or by adding a neutral buffer
solution to each of the purified modules so that their
concentrations are equivalent on a molar ratio basis. In this
example, the gel-purified vector and insert fragments have all been
adjusted to molar equivalence using the buffer 10 mM Tris, pH 8.0.
The total ligation reaction volume is set at 150 microliters. The
ligation reaction mixture consists of the following constituents:
39 microliters of ultrapure water, 15 microliters of 10.times.
Ligase buffer, 5 microliters of the purified PE3 vector backbone,
15 microliters of the purified CMV Promoter module, 15 microliters
of the purified SPC Promoter module, 15 microliters of the purified
Luciferase expression module, 15 microliters of the purified EGFP
expression module, 15 microliters of the purified SV40 3'
regulatory module, 15 microliters of the purified hGH 3' regulatory
module, and 1 microliter of ligase enzyme. The resulting reaction
components are thoroughly mixed and then incubated overnight at
16.degree. C.
[0209] The predicted vector ligation products include the
following:
pCMV-EGFP-SV40 pCMV-EGFP-hGH pCMV-Luciferase-SV40
pCMV-Luciferase-hGH pSPC-EGFP-SV40 pSPC-EGFP-hGH
pSPC-Luciferase-SV40 pSPC-Luciferase-hGH
[0210] The ligation mixture is then used to transform E. coli,
which are then spread on an LB agar plate with ampicillin. The
plate is incubated at 37.degree. C. overnight. Colonies are
isolated and propagated in individual liquid LB broth cultures. The
plasmid DNA is isolated from each LB broth culture. The DNA is
analyzed by endonuclease mapping to determine the identity of the
resulting vector incorporated into each colony.
[0211] In the preceding example, one of the predicted vector
products (pCMV-EGFP-SV40) was not produced during the first
combinatorial process. One vector that was successfully produced
(pCMV-Luciferase-SV40) can, however, serve as a vector backbone for
producing the desired pCMV-EGFP-SV40 vector. This technique can be
referred to as "Second Pass Assembly".
Example 5
Second Pass Assembly
[0212] In order to build the desired pCMV-EGFP-SV40 vector, the
pCMV-Luciferase-SV40 vector product of Example 4 is digested with
Asc I and Not I, CIP-treated, and subsequently gel-purified. This
linearized vector fragment, in which the Luciferase module has been
removed, is incubated in a ligation mixture containing the EGFP
module produced in the previous example of combinatorial vector
assembly.
[0213] The ligation mixture is used to transform E. coli, which are
then spread on an LB agar plate with ampicillin. The plate is
incubated at 37.degree. C. overnight. Colonies are isolated and
propagated in individual liquid LB broth cultures. The plasmid DNA
is isolated from each LB broth culture. The DNA is analyzed by
endonuclease mapping to determine whether the plasmids from each
colony contain the EGFP insert.
[0214] Among the many advantages of the present invention, it can
readily be appreciated that one can rapidly assemble an array of
transgenes, each containing a different combination of Promoter,
Expression, and Regulatory modules, in a very short period of time,
as well as quickly and easily vary or redesign a newly assemble
transgene. In the past, varying an assembled transgene using known
methods to create an array of different transgenes, each having
different Promoter, Expression, and Regulatory modules would
usually take a year or more of laboratory time. Using the methods
of the present invention, one can make the same number of desired
transgenes within days or weeks, and then do the desired testing of
each, thereby saving the researcher a previously large amount of
time. Further, both Dynamic Vector Assembly, in which one each of a
Promoter, Expression and Regulatory insert can be inserted into a
single backbone at the same time, and the combination method
described, in which two P-Shuttles, two E-Shuttles, and two
Regulatory-Shuttles are all combined to create eight different
types of transgenes, can be used to save precious time and money
for researchers. Shuttles that were originally created by de novo
synthesis, recombineering, and PCR terminator over-hang cloning
methods can be taken and used with the docking point technology of
the present invention to rapidly assemble these pre-made elements
into a multitude of transgenes.
[0215] While the present invention has been illustrated by the
description of embodiments thereof, and while the embodiments have
been described in detail, it is not intended to restrict or in any
way limit the scope of the appended claims to such detail.
Additional advantages and modifications will be readily apparent to
those skilled in the art. The invention in its broader aspects is
therefore not limited to the specific details, representative
methods and structures, and illustrated examples shown and
described. Accordingly, departures may be made from such details
without departing from the scope or spirit of Applicant's general
inventive concept.
Sequence CWU 1
1
1616DNAArtificial SequenceSynthetic EcoR1 endonuclease cleave site
1gaattc 626DNAArtificial SequenceSynthetic EcoR1 endonuclease
cleave site 2cttaag 6318DNAArtificial SequenceSynthetic I-Scel
endonuclease cleave site 3tagggataac agggtaat 18417DNAArtificial
SequenceSynthetic I-Scel endonuclease cleave site 4atccctattg
tccatta 17512DNAArtificial SequenceSynthetic BstX I endonuclease
cleave site 5ccannnnnnt gg 1266DNAArtificial SequenceSynthetic
BspH1 endonuclease cleaving site in pUC19 6tcatga 676DNAArtificial
SequenceMutated synthetic BspH1 endonuclease cleaving site in pUC19
7gcatga 686DNAArtificial SequenceSynthetic Acl1 endonuclease cleave
cite in pUC19 8aacgtt 696DNAArtificial SequenceMutated synthetic
Acl1 endonuclease cleave cite in pUC19 9aacgct 6106DNAArtificial
SequenceMutated synthetic Acl1 endonuclease cleave cite in pUC19
10cacgct 61111DNAArtificial SequenceSynthetic Ahd1 endonuclease
cleave site in pUC19 11gacnnnnngt c 111211DNAArtificial
SequenceMutated synthetic Ahd1 endonuclease cleave site in pUC19
12cacnnnnngt c 111334DNAArtificial SequenceSynthetic
multi-endonuclease product 13ccagataaca gggtaatatt accctgttat gtgg
341434DNAArtificial SequenceSynthetic multi-endonuclease product
14ggtctattgt cccattataa tgggacaata cacc 341514DNAArtificial
SequenceSynthetic multi-endonuclease product 15tagggataac ccta
141614DNAArtificial SequenceSynthetic multi-endonuclease product
16atcccaatag ggat 14
* * * * *