U.S. patent application number 11/907497 was filed with the patent office on 2009-08-13 for trap vectors and gene trapping using the same.
This patent application is currently assigned to TRANSGENIC INC.. Invention is credited to Kimi Araki, Ken-ichi Yamamura.
Application Number | 20090202988 11/907497 |
Document ID | / |
Family ID | 16433801 |
Filed Date | 2009-08-13 |
United States Patent
Application |
20090202988 |
Kind Code |
A1 |
Yamamura; Ken-ichi ; et
al. |
August 13, 2009 |
Trap vectors and gene trapping using the same
Abstract
A trap vector containing a loxP sequence composed of inverted
repeat sequence 1, a spacer sequence and inverted repeat sequence 2
in this order, the loxP sequence being a mutant loxP wherein a part
of the inverted repeat sequence 1 or 2 is mutated.
Inventors: |
Yamamura; Ken-ichi;
(Kumamoto-shi, JP) ; Araki; Kimi; (Kumamoto-shi,
JP) |
Correspondence
Address: |
BIRCH STEWART KOLASCH & BIRCH
PO BOX 747
FALLS CHURCH
VA
22040-0747
US
|
Assignee: |
TRANSGENIC INC.
Kumamoto-shi
JP
|
Family ID: |
16433801 |
Appl. No.: |
11/907497 |
Filed: |
October 12, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10030658 |
Jan 11, 2002 |
7312075 |
|
|
PCT/JP00/02916 |
May 2, 2000 |
|
|
|
11907497 |
|
|
|
|
Current U.S.
Class: |
435/6.16 ;
435/320.1; 435/325 |
Current CPC
Class: |
C12N 15/1051 20130101;
C12N 15/10 20130101 |
Class at
Publication: |
435/6 ;
435/320.1; 435/325 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12N 15/00 20060101 C12N015/00; C12N 5/06 20060101
C12N005/06 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 14, 1999 |
JP |
JP 1999-200997 |
Claims
1. A trap vector comprising a loxP sequence and a lox71 sequence,
wherein the loxP sequence comprises in sequential order inverted
repeat sequence 1, a spacer sequence, and inverted repeat sequence
2; wherein the lox71 sequence comprises a nucleotide sequence shown
in SEQ ID NO:15; and wherein the trap vector is suitable for use in
mammalian cells.
2. A trap vector comprising a loxP sequence and a lox66 sequence,
wherein the loxP sequence comprises in sequential order inverted
repeat sequence 1, a spacer sequence, and inverted repeat sequence
2; wherein the lox66 comprises a nucleotide sequence shown in SEQ
ID NO: 16; and wherein the trap vector is suitable for use in
mammalian cells.
3. A vector generated from recombination between: (a) a trap vector
comprising a loxP sequence and a mutant loxP sequence, wherein the
loxP sequence comprises in sequential order inverted repeat
sequence 1, a spacer sequence, and inverted repeat sequence 2;
wherein the mutant loxP sequence comprises a sequence of which a
part of said inverted repeat sequence 1 of loxP is mutated; and (b)
a trap vector comprising a mutant loxP sequence, wherein the mutant
loxP sequence comprises in sequential order inverted repeat
sequence 1, a spacer sequence, and inverted repeat sequence 2;
wherein a part of said inverted repeat sequence 2 is mutated, or
(c) a trap vector comprising a loxP sequence and a mutant loxP
sequence, wherein the loxP sequence comprises in sequential order
inverted repeat sequence 1, a spacer sequence and inverted repeat
sequence 2; wherein the mutant loxP sequence comprises a sequence
of which a part of said inverted repeat sequence 2 of loxP is
mutated; and (d) a trap vector comprising a mutant loxP sequence,
wherein the mutant loxP sequence comprises in sequential order
inverted repeat sequence 1, a spacer sequence and inverted repeat
sequence 2; wherein a part of said inverted repeat sequence 1 is
mutated, wherein recombination of the mutant loxP occurs more
efficiently than the reverse reaction as compared to wild-type
loxP.
4. The vector of claim 3, wherein said vector does not undergo
recombination with another loxP.
5. A method of gene trapping, comprising the steps of: introducing
the trap vector of claim 1 or 2 into embryonic stem cells;
culturing the embryonic stem cells; selecting those cells which
exhibit a pattern of single copy integration of the trap vector;
and isolating the trapped gene.
6. Embryonic stern cells into which the trap vector of claim 1 or 2
is introduced.
7. A method of gene trapping, said method comprising the steps of:
introducing into embryonic stem cells: (a) a trap vector comprising
a loxP sequence, a marker gene and a mutant loxP sequence, wherein
the loxP sequence comprises in sequential order inverted repeat
sequence 1, a spacer sequence, and inverted repeat sequence 2;
wherein the mutant loxP sequence comprises a sequence of which a
part of said inverted repeat sequence 1 of loxP is mutated, and (b)
a trap vector comprising a mutant loxP sequence, wherein the mutant
loxP sequence comprises in sequential order inverted repeat
sequence 1, a spacer sequence, and inverted repeat sequence 2;
wherein a part of said inverted repeat sequence 2 is mutated, or
(c) a trap vector comprising a loxP sequence, a marker gene and a
mutant loxP sequence, wherein the loxP sequence comprises in
sequential order inverted repeat sequence 1, a spacer sequence and
inverted repeat sequence 2; wherein the mutant loxP sequence
comprises a sequence of which a part of said inverted repeat
sequence 2 of loxP is mutated; and (d) a trap vector comprising a
mutant loxP sequence, wherein the mutant loxP sequence comprises in
sequential order inverted repeat sequence 1, a spacer sequence and
inverted repeat sequence 2; wherein a part of said inverted repeat
sequence 1 is mutated, wherein recombination of the mutant loxP
occurs more efficiently than the reverse reaction as compared to
wild-type loxP, and wherein trap vector (a) or (b) further
comprises at least one splice acceptor site and at least one
internal ribosomal entry site; culturing the embryonic stem cells;
selecting those cells which exhibit a pattern of single copy
integration of the trap vector; and isolating the trapped gene.
8. The method according to claim 7, wherein the trap vector further
comprises pA and pV, wherein pA is located downstream of the marker
gene.
Description
[0001] This application is a Divisional of co-pending application
Ser. No. 10/030,658 filed on Jan. 11, 2002 and for which priority
is claimed under 35 U.S.C. .sctn. 120. Application Ser. No.
10/030,658 is the national phase of PCT International Application
No. PCT/JP00/02916 filed on May 2, 2000 under 35 U.S.C. .sctn. 371.
The entire contents of each of the above-identified applications
are hereby incorporated by reference.
TECHNICAL FIELD
[0002] The present invention relates to random mutation ES clone
technology using gene trapping.
BACKGROUND ART
[0003] It is said that structural analysis of human genome will be
completed in or before 2003 as the human genome project is
progressing well. Now, the age of isolating genes one by one and
analyzing their structures separately seems to be over, and we have
come into the age of "structural analysis" of genome.
[0004] With the nucleotide sequence of genome alone, however,
information on functions is insufficient. Thus, a novel analysis
system for functional analysis is needed. Further, although one of
the major goals of human genome analysis is to elucidate causative
genes in human diseases, such diseases cannot be explained with the
structures of causative genes alone.
[0005] Accordingly, production of model individuals is an
indispensable assignment in order to analyze processes of disease
development and to develop new treatment methods after the
identification of causative genes.
[0006] On the other hand, if genome is divided into gene regions
and non-gene regions in terms of structure, it is considered that
these two parts have separate functions and that it is necessary to
analyze the functions of both parts (FIG. 1). From the viewpoint of
entire genome, each gene is performing only a part of the entire
function. Genome is not a mere collection of genes and may have
unknown functions. In fact, a new concept "position effect
mutation" has been established. From this, it is presumed that
genome has regions of unknown functions.
[0007] Gene regions are composed of regulatory regions and coding
regions. At present, the target of genome functional analysis is
coding regions. When mouse is compared with human, the kinds of
genes they have are almost equal. Therefore, functional analysis of
the regulatory region is important. There is difference in species
between mouse genes and human genes. It is believed that this
difference is not due to difference in protein but due to
difference in the regulation of gene expression.
[0008] The function of a transcription factor or the like involved
in the regulation of gene expression can be elucidated from the
sequence of the coding region of the relevant gene. The analysis of
the functions of those elements to which the transcription factor
binds is extremely difficult at present because a number of those
elements exist in the regulatory region of one gene. However, as a
technique of functional analysis, a method using bacterial
artificial chromosomes may be considered.
[0009] It is considered that functional analysis of coding regions
may be performed at the mRNA level, protein level, cell level,
tissue/organ level and individual (i.e. whole animal) level. It is
believed that such analysis at the mRNA level can be performed
using DNA chips. On the other hand, the use of embryonic stem (ES)
cells seems to be the best way for performing functional analysis
at other levels, because various cell and tissue derivative systems
have been developed directly from ES cells in vitro and a number of
such systems are expected to be developed in the future.
Furthermore, the use of ES cells is advantageous in that individual
level analysis systems can be established.
[0010] From the foregoing, it is understood that gene knockout at
ES cell level and production of knockout mice in which the relevant
genes are knocked out are extremely important in functional
analysis of genome.
[0011] To date, homologous recombination using ES cells has played
a major role in the production of knockout mice. However,
considering this method not as a strategy of producing knockout
mice separately but from a strategic viewpoint of producing
knockout mice comprehensively, this method has serious
problems.
[0012] First, this method requires too much time. In the production
of knockout mice, it is the rate-determining step to isolate
knockout ES clones generated through homologous recombination using
ES cells. Even a skillful researcher needs at least three months
for isolating a knockout ES clone. Thus, only four genes can be
knocked out in one year. Accordingly, in the case of introducing
each one mutation into 10.sup.5 genes, 2,500 researchers are
required for one year. It is estimated that approximately 1,000
lines of knockout mice are produced in one year in the world. This
means that it would take 100 years to produce 10.sup.5 knockout ES
clones. This is so unrealistic compared to the advance in the
structural analysis of human genome that is to be completed in
2003.
[0013] Secondly, this method requires too much cost. At least 2 to
4 million yen is necessary to produce one line of knockout mouse
excluding personnel expenses and depreciation expenses. Thus,
production of 10.sup.5 simple knockout mice requires 200 to 400
billion yen.
[0014] As described above, the conventional homologous
recombination using ES cells has problems, and genome is vast.
However, the number of genes in genome is limited. Thus, it is
necessary to isolate from genome those genes having important
functions. In many cases, the function of a gene is elucidated only
after production of a knockout mouse in which the relevant gene is
disrupted. Therefore, knockout mice are directly connected with
future development of epoch-making drugs and have extremely high
value added. Under circumstances, it has become the world's
"strategy" to produce mutant mice at random and in large scale. At
present, the three methods described below are considered most
reasonable in the production of random mutation mice.
[0015] The first one is a method using ethylnitrosourea (ENU), a
mutagen. A project of large-scale mutant production using ENU has
been started in Europe. In Germany, Dr. Balling of the Institute of
Mammalian Genetics and others started this project in 1997 as a
part of the human genome project. In England, supported by
SmithKline Beecham, Dr. Brown and others started this project at
MRC Mouse Genome Center in Harwell aiming at establishment of
mutant mice having mutations mainly in brain/nervous system. To
date, these two groups have established approximately 200 lines of
mutant mice exhibiting dominant inheritance. The project is
proceeding more efficiently than expected. In the United States, it
has been decided that structural analysis of mouse genome and
production of mutant by the ENU method start with a huge budget (6
billion yen/year) at Case Western Reserve University, Oak Ridge
National Laboratory, etc.
[0016] When ENU is administered to adult male mice, ENU acts on
spermatogoniums before meiosis and causes about 50 to 100 point
mutations per spermaogonium at random. Mutations occur at a
frequency of 1/1,000/gamete per locus. Therefore, by crossing one
treated male mouse with one normal female mouse, many kinds of
mutant mice can be produced in F1 generation. In the method using
ENU, if 1,000 mice are screened for a specific locus, one mouse has
a mutation caused in that locus in terms of probability. Thus, this
method is considered highly efficient.
[0017] The second method is a method using chlorambucil that is
also a mutagen. This method causes mutations in spermatogoniums at
the same frequency as in the method using ENU. However, these
mutations are deletion mutations, and sometimes as many as one
megabases may be deleted.
[0018] The third method is a method using gene trapping. Gene
trapping is a technique that was developed for the purpose of
searching for unknown genes by introducing trap vectors containing
a marker gene into ES cells and then monitoring the expression of
the marker gene. Trap vectors are integrated into ES cells at
random and, as a result of their integration, endogenous genes
(genes present in cells and tissues inherently) are disrupted in
most cases. Therefore, preparing chimeric mice from such ES cells
can produce various knockout mice.
[0019] However, each of the methods using a mutagen and the method
using gene trapping has an advantage(s) and a drawback(s) (Table
1).
TABLE-US-00001 TABLE 1 ENU Chlorambucil Gene Method Method Trapping
Nature of Mutation Point Deletion Any desired mutation mutation
mutation Production of Mutant Easy Easy Difficult Mouse
Identification of Difficult Medium Easy Mutant Mouse Other Features
Can use ES trap clones
[0020] According to the ENU method, production of mutant mice is
easy, but establishment of individual mutant lines is not easy
because segregation by crossing should be conducted. Further, in
order to identify mutated genes, the relevant locus should be
identified first by linkage analysis using polymorphic DNA markers,
and then the gene should be isolated by positional cloning. Thus,
the ENU method requires complicated operations.
[0021] According to the chlorambucil method, production of mutant
mice is easy, but deleted sites should be identified. For that
purpose, analysis must be made using a number of polymorphic DNA
markers. Besides, generally, methods using a mutagen such as
chlorambucil need large breeding rooms. Thus, such methods require
much expenses and labor.
[0022] Although the gene trap method requires labor and technology
for producing mutant mice, identification of mutated genes is easy
and experiments can be conducted according to the size of breeding
rooms. Gene trap ES clones per se are precious resource for
functional analysis of genome. The gene trap method is also
remarkably different from other methods in this point.
[0023] Some laboratories in the world have already started
production of mutants by gene trapping. In the United States, a
private firm Lexicon Genetics Incorporated is undertaking random
disruption by gene trapping using retrovirus vectors. However,
ordinary researchers can hardly use this service because of the
following reasons. Briefly, it is not sure whether an endogenous
gene is disrupted or not even if the gene is trapped; it is not
clear whether germline chimeric mice can be produced; an additional
charge is required for the production of chimeric mice; and
considerable charges are required for using the service. In
Germany, gene trapping is performed toward a goal of 12,000 clones
as a part of the ENU project. Anyway, these are proceeding focusing
on the analysis of trapped genes rather than the establishment of
mouse lines.
DISCLOSURE OF THE INVENTION
[0024] The problem for solution by the invention is to overcome the
problems that conventional gene trap methods have, to develop a
novel "exchangeable gene trap method" that seems almost ideal, to
establish ES trap clones in large scale using the above method, and
to produce mouse mutants using the trap clones. Thus, it is an
object of the invention to provide trap vectors; a method of gene
trapping; transgenic or knockout animals in which a trapped gene is
introduced; and trapped genes.
[0025] As a result of intensive and extensive researches toward the
solution of the above problems, the present inventors have reached
an idea of using the bacteriophage-derived recombination system
Cre-loxP in gene trapping. Thus, the present invention has been
achieved. Cre is a recombinase that recognizes a loxP sequence and
causes recombination at that site.
[0026] The present patent application provides the following
inventions:
(1) A trap vector containing a loxP sequence composed of inverted
repeat sequence 1, a spacer sequence and inverted repeat sequence 2
in this order, the loxP sequence being a mutant loxP wherein a part
of inverted repeat sequence 1 or 2 is mutated.
[0027] As a specific example of the mutant loxP in which a part of
its inverted repeat sequence 1 is mutated, lox71 (e.g. the sequence
shown in SEQ ID NO: 1) may be given. As a specific example of the
mutant loxP in which a part of its inverted repeat sequence 2 is
mutated, lox66 (e.g. the sequence shown in SEQ ID NO: 2) may be
given.
(2) A vector generated from recombination between a trap vector
containing a mutant loxP wherein a part of inverted repeat sequence
1 is mutated and a the trap vector containing a mutant loxP wherein
a part of inverted repeat sequence 2 is mutated. (3) A trap vector
selected from the group consisting of the following (a) to (i):
[0028] (a) SD-SA-lox71-IRES-M-loxP-PV-SP
[0029] (b) SP-lox71-IRES-M-loxP-PV-SP
[0030] (c) SA-lox71-IRES-M-loxP-pA-PV-SP
[0031] (d) SA-lox71-IRES-M-loxP-puro-pA-PV-SP
[0032] (e) lox71-M-loxP-pA-lox2272-PV-lox511
[0033] (t) lox71-IRES-M-loxP-pA-lox2272-PV-lox511
[0034] (g) (lox71-integrated SA)-M-loxP-pA-lox2272-PV-lox511
[0035] (h) (lox71-integrated
SA)-IRES-M-loxP-pA-lox2272-PV-lox511
[0036] (i) (lox71-integrated
SA)-M-loxP-pA-lox2272-promoter-M-lox511-SD,
wherein SP represents any sequence; SA represents a splice
acceptor; SD represents a splice donor; IRES represents an internal
ribosomal entry site; M represents a marker gene; puro represents
puromycin resistance gene ; pA represents a poly(A) sequence; and
PV represents a plasmid vector.
[0037] In the trap vectors (a) to (i) described above, .beta.-geo
gene may be given as a specific example of the marker gene, and
pBR322, pUC plasmids (pUC18, pUC19, pUC118, pUC119, etc.), pSP
plasmids (pSP64, pSP65, pSP73, etc.) and pGEM plasmids (pGEM-3,
pGEM-4, pGEM-3Z, etc.) may be enumerated as specific examples of
the plasmid vector.
(4) A method of gene trapping comprising introducing any of the
above-described trap vectors into embryonic stem cells, and
embryonic stem cells into which the trap vector is introduced by
the method. (5) A method for producing a transgenic animal or
knockout animal comprising introducing the above-described
embryonic stem cells into an animal, and a transgenic animal or
knockout animal produced by the method.
[0038] As a specific example of the above animal, one selected from
the group consisting of mouse, rat, rabbit, guinea pig, pig, sheep
and goat may be given.
[0039] Hereinbelow, the present invention will be described in
detail. The present specification encompasses the contents of the
specification and/or drawings of the Japanese Patent Application
No. 11-200997 based on which the present application claims
priority.
[0040] The present invention relates to a method of gene trapping,
transgenic or knockout animals into which a trapped gene is
introduced, and trapped genes. An outline of the method of gene
trapping according to the invention is shown in FIG. 2. First, in
order to achieve the object of the present invention, a trap vector
is constructed and introduced into ES cells, followed by isolation
and selection of trap clones (FIG. 2A-C). In FIG. 2, pU-Hachi
vector is exemplified. Subsequently, chimeric animals (e.g.
chimeric mice) are produced, followed by production of mutant mice
derived from the trap clone (FIG. 2F-G). On the other hand, using
the trapped and selected clone, isolation and sequencing of the
trapped gene as well as recovery of the genome by plasmid rescue
are performed (FIG. 2C-E). Further, the clone is subjected to
electroporation and selection with a drug such as puromycin to
thereby trap a gene of interest. Then, the trapped gene is
expressed, followed by production of ES clone-derived mouse lines
(FIG. 2H-I).
[0041] The present invention can be summarized as follows
(including pilot studies).
(l) Overall Efficiency
(1-1) Screening by Formation of Embryoid Bodies
[0042] One hundred and six neomycin resistant clones were
suspension-cultured for the formation of embryoid bodies. The
expression of .beta.-galactosidase was analyzed at the stage of ES
cells and after the induction of differentiation. As a result, it
was found that 90 trap clones (86%) were expressing
.beta.-galactosidase at any one of the above stages.
(1-2) Selection of Clones Indicating Single Copy Integration
[0043] DNA was extracted from 109 trap clones that had expressed
the marker gene during the process of embryoid body formation, and
then integration patterns of the trap vector were analyzed. As a
result, 75 clones (70%) had a single copy integrated. Of these, 24
clones (22%) were complete (i.e. retained the replication origin of
the plasmid) and 40 clones (37%) lacked pUC. Even if pUC was lost,
it could be re-inserted by using lox71 site. Therefore, these 64
clones (59%) were found to be useful (Table 4).
(1-3) Efficiency of Germline Chimera Production
[0044] Chimeric mice were produced using the above-mentioned trap
clones. As a result, germline chimeric mice were obtained from
approximately one half of the clones.
(1 -4) Summary of the Entire Experiment
[0045] It was found that about 26% of the neomycin resistant clones
selected initially reached the final stage of the experiment. Since
the efficiency of germline chimera production is now increasing, it
is believed that the overall efficiency can be increased further.
However, the efficiency achieved at this time seems to be
sufficient for the practice of researches.
(2) Efficiency of the Gene Trap Method
[0046] As a result of the tests so far conducted, 24 trap lined
were established. Of these, 13 lines have proceeded to analysis at
the gene level. Nucleotide sequences of these lines were compared
with GenBank and EMBL databases using BLAST program. The results
were as follows: 9 clones individually trapped a known gene; 3
clones individually trapped an EST; and the remaining 1 clone
trapped an unknown gene (Table 2). According to the reports so far
made by other researchers, 10-25% of trapped genes are known genes;
10-20% are ESTs; 50-80% are unknown genes; and 2-10% are
repeats.
TABLE-US-00002 TABLE 2 Known Gene EST Unknown Gene Repeat Present 9
(69.2%) 3 (23.1%) 1 (7.7%) Invention Previous 10-25% 10-20% 50-80%
2-10% Reports
(3) Trapped Genes
[0047] The inventors examined whether those genes involved in
development and cell growth had been efficiently trapped or not by
ascertaining the kinds of known genes by a screening method
utilizing formation of embryoid bodies. As a result, it was found
that the known genes were CBP (CREB binding protein) and Sp1 that
are transcription factors; cyclin B2 involved in cell cycle; Crk
and pHPS1-2 involved in signal transduction; rRNA, suil, hnRNP L
and RNA polymerase I; and mitochondrial DNA (Table 3). Thus, it was
found that very common genes were trapped. A major part of these
genes are involved in cell growth. This suggests that the screening
system utilizing formation of embryoid bodies works well.
TABLE-US-00003 TABLE 3 Class Clone No. Gene 1. Nucleus (1)
Transcription Ayu3-112 CBP Ayu8-038 Sp1 (2) Cell Cycle Ayu3-008
Cyclin B2 Ayu6-003 Homologous to the E. coli cell division protein
Ftsj1 (3) Signal Ayu8-104 Crk Transduction Ayu8-025 pHPS.sub.1-2
(4) Cell Skeleton Ayu8-003 dynamin II 2. Cytoplasm (1) Translation
Ayu3-022 rRNA Ayu8-016 sul1 Ayu8-016 Upstream region of hnRNP L
Ayu8-019 Very likely to be RNA polymerase I (2) Others Ayu3-001
Mitochondrial DNA 3. Unknown Ayu7-003 Unknown
(4) Confirmation of Gene Disruption by Trapping
[0048] It is one of the major points whether endogenous genes have
been actually disrupted or not by gene trapping. Thus, the
inventors have analyzed the structure of the trap site for 6 known
genes. As a result, it was found that the trap vector was inserted
into the promoter region in one gene; into an exon in one gene; and
into an intron in 4 genes. In all of them, the gene was completely
or partially disrupted. Therefore, it has become clear that
endogenous genes can be disrupted efficiently by the method of gene
trapping of the invention (FIG. 10).
[0049] Hereinbelow, the present invention will be described in more
detail.
1. Construction of Trap Vectors
[0050] Gene trapping is a method for trapping unknown genes on
genome utilizing the fact that trap vectors introduced into ES
cells are integrated into mouse endogenous genes incidentally and
at random. "Gene trapping" means that a trap vector enters into a
specific gene on genome and captures that gene. The vector for gene
capturing is called "trap vector". Genes have enhancers, promoters,
exons, poly(A) sequences, etc. The trap vector is capable of
capturing any of them. For this purpose, a trap vector with a
structure suitable for the specific capturing may be used.
[0051] Generally, exon trap vectors are composed of a reporter gene
with a splice acceptor alone, a drug selection marker gene and a
plasmid. Only when these vectors are integrated downstream of a
mouse endogenous gene, the reporter gene is expressed. This means
that it is possible to know the vector's integration into an
endogenous gene by monitoring the expression of the reporter gene
in the trap vector. If a plasmid such as pUC19 has been linked to
the trap vector, the trapped endogenous gene can be isolated by the
technique called plasmid rescue. "Plasmid rescue" is a technique
for recovering a gene of interest by selection with ampicillin,
etc. of those cells transformed with electroporation or the like
(FIG. 2E). Furthermore, since the endogenous gene is disrupted at
the time of trapping, knockout mice can be produced immediately.
Further, since the reporter gene is expressed under the control of
the expression regulatory region of the endogenous gene, the tissue
specificity and time specificity of the gene can be analyzed
easily.
[0052] In conventional gene trapping methods, even if a mouse
endogenous gene could be disrupted completely, it has been
impossible to introduce thereinto subtle mutations, such as single
amino acid substitution, seen in human hereditary diseases. Also,
it has been impossible to replace the disrupted mouse gene with a
human gene. Toward the solution of these problems, the present
invention has modified the Cre-loxP system (a bacteriophage-derived
recombination system) and utilized it in the trap vector in gene
trapping. As a result, it has become possible to insert any gene
into a mutant loxP site of the trap vector after a mouse gene has
been disrupted as a result of the integration of the trap vector.
According to the present invention, it has become possible to
introduce subtle mutations, such as single amino acid substitution,
seen in human hereditary diseases. It has also become possible to
replace the trapped gene with a human gene. The trap vector of the
invention may be used for trapping various genes. In particular, it
may be used preferably for exon trapping or promoter trapping.
[0053] loxP (locus of crossing (X-ing) over, P1) is a 34 bp
sequence (5'-ataacttcgtata gcatacat tatacgaagttat-3') (SEQ ID NO:
3). The 13 bases at its 5' end (called "inverted repeat sequence
1") and the 13 bases at its 3' end (called "inverted repeat
sequence 2") constitute inverted repeat sequences, which are
separated by an 8 bp spacer (gcatacat) (FIG. 3). The term "inverted
repeat sequences" used herein means that a sequence located on one
side of the spacer is complementary to a sequence located on the
other side of the spacer in opposite orientation. In other words,
the sense strand of one sequence is homologous to the antisense
strand of the other sequence in opposite orientation to each other.
These two repeat sequences have opposite orientation and, when a
double-strand is formed, one same sequence is repeated. Thus, they
are called inverted repeat sequences. As shown in FIG. 3, in one
strand (for example, the sense strand) of the double-strand,
inverted repeat sequence 1 (5'-ataacttcgtata-3'; SEQ ID NO: 4) (the
left side in FIG. 3) is complementary, in the 5'.fwdarw.3'
direction, to inverted repeat sequence 2 (5'-tatacgaagttat-3'; SEQ
ID NO: 5) (the right side in FIG. 3) in the 3'.rarw.5'
direction.
[0054] Unlike ordinary sequences, loxP has directionality.
Therefore, when the loxP sequence is represented in the
above-mentioned 5'.fwdarw.3' direction in the present invention, an
arrow pointing the left (e.g. will be included in the
representation.
[0055] Cre (causes recombination) means a recombinase that causes
genetic recombination and, upon recognition of the above-described
repeats, cleaves the spacer in such a manner that "cataca" in the
spacer is left as a cohesive end (FIG. 3).
[0056] In bacteria, recombination occurs between two loxP sites,
and insertion or deletion reaction takes place. If it is possible
to cause insertion reaction in mammal cells, then any desired gene
can be inserted later. This would dramatically expand the
applicability of gene trapping. Actually, since mammal cells have
large nuclei, circular DNA molecules with once deleted loxP will
diffuse and insertion reaction is hardly observed.
[0057] Toward the solution of the above problems, the present
inventors have elaborated a method in which mutations are
introduced into the loxP sequence in order to cause insertion
reaction and, once a gene has been inserted into genome, the gene
does not undergo deletion (i.e. not removed from the genome). For
this method, the inventors have prepared two mutant loxP sequences
(FIG. 4).
[0058] Briefly, the inventors created one mutant by introducing
substitution mutations into one of the inverted repeat sequences of
loxP (sense strand)(ATAACTTCGTATA (SEQ ID NO: 4); shown at the left
in FIG. 4b) so that the mutated sequence becomes TACCGTTCGTATA
(underlined portion was changed). This mutant is designated "lox71"
(SEQ ID NO: 1; FIG. 4b). The other mutant was created by
introducing substitution mutations into the other inverted repeat
sequence of loxP (sense strand)(TATACGAAGTTAT (SEQ ID NO: 5); shown
at the right in FIG. 4b) so that the mutated sequence becomes
TATACGAACGGTA (underlined portion was changed). This mutant is
designated "lox66" (SEQ ID NO: 2; FIG. 4b).
[0059] When recombination has occurred between lox71 on genome and
lox66 on a plasmid, a loxP sequence having mutations in both
repeats (designated "lox71/66"; TACCGTTCGTATA GCATACAT
TATACGAACGGTA; SEQ ID NO:6) is located on the 5' side of the
inserted DNA (FIG. 4a, see at the left) and a wild-type loxP
sequence (ATAACTTCGTATA GCATACAT TATACGAAGTTAT; SEQ ID NO: 3) on
the 3' side of the inserted DNA (FIG. 4a, see at the left). As a
result, Cre no longer can recognize lox71/66 and thus cannot cause
recombination with loxP. In the case of homologous recombination
between two wild-type loxP sequences, a circular DNA containing the
excised loxP is physically separated. Thus, the reaction tends
toward deletion rather than insertion. On the other hand, when
lox71 is used on chromosomes and lox66 is used on circular DNA
molecules, Cre has difficulty in recognizing the lox71/66 generated
as a result of integration of the DNA. Thus, the reaction tends
toward insertion rather than deletion, and the inserted state of
insertion is maintained (FIG. 5). It should be noted that, in the
present invention, lox66 may be used on chromosomes, and lox71 may
be used on circular DNA molecules.
[0060] Actually, when a mutant loxP (hereinafter, sometimes
referred to as "mutant lox") such as lox71 has been integrated into
ES cells in advance, and a plasmid containing other mutant loxP
(e.g. lox66) is introduced thereinto, the plasmid is integrated
into the genome. Therefore, if this lox71, for example, has been
integrated into a gene trap vector in advance, it becomes possible
to insert any desired gene later by using lox66. Thus, according to
the present invention, it has become possible to replace the
trapped gene with a gene into which a subtle mutation(s) has (have)
been introduced or a human gene.
[0061] Gene trap vectors using this mutant lox (lox71 or lox66) may
be constructed as described below (see FIG. 6). Here, it should be
noted that the following trap vectors are provided only for
illustration, not for limitation. Thus, although lox71 is used as
an example of a mutant lox in the following vectors, vectors using
lox66 instead of lox71 are also included in the present
invention.
[0062] (a) U8: SP-SA-lox71-IRES-M-pA-loxP-PV-SP
[0063] (b) U8delta: SP-lox71-IRES-M-pA-loxP-PV-SP
[0064] (c) pU-Hachi: SA-lox71-IRES-M-loxP-pA-PV-SP
[0065] (d) pU-12: SA-lox71-IRES-M-loxP-puro-pA-PV-SP
[0066] (e) pU-15: lox71-M-loxP-pA-lox2272-PV-lox511
[0067] (f) pU-16: lox71-IRES-M-loxP-pA-lox2272-PV-lox511
[0068] (g) pU-17: (lox71-integrated
SA)-M-loxP-pA-lox2272-PV-lox511
[0069] (h) pU-18: (lox71-integrated
SA)-IRES-M-loxP-pA-lox2272-PV-lox511
[0070] (i) (lox71-integrated
SA)-M-loxP-pA-lox2272-promoter-M-lox511-SD
[0071] In the above-described vector components, SP represents any
sequence; SA represents a splice acceptor; SD represents a splice
donor; IRES represents an internal ribosomal entry site; M
represents a marker gene; puro represents puromycin resistance
gene; pA represents a poly(A) sequence; and PV represents a plasmid
vector.
[0072] When trap vectors are integrated into genomic DNA, a part of
the vector is deleted in most cases and, as a result, an important
part of the vector may be missed. SP is any sequence added as a
dummy to prevent such deletion. This sequence may be selected at
one's discretion. The length of SP is 100-1000 bp, preferably
300-400 bp. Any known sequence may be used as SP. For example, a
part of rabbit .beta.-globin gene may be used.
[0073] The splice acceptor means a sequence that can be linked to
the 3' end of an exon at the time of splicing.
[0074] The splice donor means a sequence that can be linked to the
5' end of an exon at the time of splicing.
[0075] IRES, called "internal ribosomal entry site", is a site on
ribosome to which aminoacyl t-RNA is bound during protein synthesis
(A site), and it is a sequence to allow translation to start in a
CAP independent manner.
[0076] The marker gene is a gene that serves as a marker to
indicate whether the vector of the invention could trap a target
gene. Specific examples of marker genes include E. coli-derived
.beta.-galactosidase gene (lacZ gene) or a fusion gene between lacZ
gene and neomycin (G418) resistance gene (.beta.-geo gene), CAT
gene, GFP gene, SV40 large T gene, neomycin resistance gene,
puromycin resistance gene, hygromycin resistance gene, and
blasticidin resistance gene.
[0077] The plasmid vector is used after gene trapping to isolate
the endogenous gene by plasmid rescue. Plasmid rescue technique is
a method for recovering adjacent regions of the plasmid (which is
replicable in E. coli) integrated in a trap vector using a part of
the plasmid. For example, when a genomic DNA segment is linked to
the plasmid, a fragment consisting of the plasmid and the genomic
DNA segment linked thereto is excised by restriction enzyme
treatment. The excised fragment is made circular and introduced
into E. coli, which is then propagated. As a result, the genomic
DNA segment flanking the plasmid can be recovered. Specific
examples of the plasmid vector include pBR322, pUC (pUC18, pUC19,
pUC118, pUC119, etc.), pSP (pSP64, pSP65, etc.), and pGEM (pGEM-3,
pGEM-4, pGEM3Z, etc.). In addition, supF, ampicillin resistance
gene, origin of replication, or restriction sites for cloning (e.g.
multicloning site) may be linked to the plasmid vector
independently or in an appropriate combination.
[0078] The vector shown in (a) above is designated "U8". The basic
part of U8 (SA-IRES-.beta.-geo-pA; FIG. 7) is derived from
pGT1.8IRESbetageo. This pGT1.8IRESbetageo contains mouse En-2
gene-derived splice acceptor, IRES and .beta.-geo. lox71 is
inserted into the BglII site of this plasmid followed by SalI
treatment to thereby provide a SalI fragment. On the other hand,
plasmid pEBN-Seti is prepared by inserting into a vector (such as
pUC19) a 180 bp SP sequence, loxP and poly(A) signal. The SalI
fragment obtained above is inserted into the SalI site of this
plasmid to produce U8. Thus, the structure of this trap vector is
expressed as follows (from the 5' end, in this order): any
sequence, splice acceptor, lox71, IRES, .beta.-geo, pA, loxP,
pUC19, and any sequence (FIG. 7).
[0079] The vector shown in (b) above is designated "U8delta".
U8delta is obtainable by deleting the splice acceptor from U8. This
vector has a structure in which lox71 is linked before the reporter
.beta.-geo and loxP after .beta.-geo. This vector was given such a
structure because the intermediate IRES and .beta.-geo can be
removed completely by transiently expressing Cre after the vector
has been integrated. As a result, plasmid pUC is located close to
the mouse endogenous gene which was located upstream of the
plasmid. Thus, the mouse endogenous gene can be isolated
easily.
[0080] The vector shown in (c) above is designated "pU-Hachi". This
vector is composed of SA-lox71-IRES-M-loxP-pA-PV-SP. pU-Hachi
vector is derived from pGT1.8IRES .beta.-geo, and contains SA
sequence from mouse En-2 gene and .beta.-geo sequence linked to
encephalomyocarditis virus-derived IRES sequence. A BamHI fragment
of lox71 is inserted into the BglII site of pGT1.8IRES .beta.-geo.
Then, a plasmid was constructed by inserting an SP sequence, a loxP
sequence, and poly A addition signal from mouse phosphoglycerate
kinase-1 (PGK) into a modified vector from which lacZ sequence has
been removed. The SP sequence is used to protect the 3' end of the
trap vector. pU-Hachi is obtainable by inserting a SalI fragment of
SA-IRES-lox71-.beta.-geo into the SalI site of the above
plasmid.
[0081] The vector shown in (d) above is designated "pU-12". This
vector is composed of SA-lox71-IRES-M-loxP-puro-pA-PV-SP. In order
to construct this pU-12 trap vector, first, the PGK poly(A) signal
of pE3NSE7 is replaced with puromycin resistance gene+PGK poly(A)
signal. Then, lox511 is inserted into the BglII site downstream
thereof. Then, a SalI fragment of SA-IRES-lox71-.beta.-geo from
pU-Hachi is inserted into the restriction site of the resultant
plasmid to thereby obtain pU-12.
[0082] The vector shown in (e) above is designated "pU-15". This
vector is composed of lox71-M-loxP-pA-lox2272-PV-lox511. "lox2272"
is a mutant loxP in which the spacer sequence (gcatacat) is changed
to ggatactt (i.e. the second base "c" has been changed to "g", and
the seventh base "a" to "t"). "lox511" is a mutant loxP in which
the spacer sequence (gcatacat) is changed to gtatacat (i.e. the
second base "c" has been changed to "t"). Since lox511 and lox2272
have mutations in the spacer, they do not cause recombination with
other loxP sequences such as wild-type loxP or lox71, though two
lox511 sequences or lox2272 sequences cause recombination with each
other. The order of lox2272 and lox511 in the vector may be
changed. Either one may come first. (This will apply to other
vectors using these mutants.)
[0083] The vector shown in (f) above is designated "pU-16". This
vector is composed of lox71-IRES-M-loxP-pA-lox2272-PV-lox511, and
is obtainable by inserting IRES between lox71 and .beta.-geo of
pU-15.
[0084] The vector shown in (g) above is designated "pU-17". In this
vector, lox71 is integrated in a region of SA. This vector may be
constructed as follows. Briefly, a plasmid is constructed by
inserting lox511, loxP, PGK poly(A) signal and lox2272 into, for
example, pSP plasmid. Then, lox71 is inserted into SA in pU-Hachi
followed by insertion of .beta.-geo in this order. This plasmid is
ligated to the plasmid constructed above to thereby obtain
pU-17.
[0085] The vector shown in (h) is designated "pU-18". Like pU-17,
this vector also has lox71 integrated in SA. pU-18 is obtainable by
inserting IRES between SA and .beta.-geo of pU-17.
[0086] The vector shown in (i) is composed of (lox71-integrated
SA)-M-loxP-pA-lox2272-promoter-M-lox511-SD. This vector is
obtainable by inserting a promoter and M in this order into pU-17
instead of PV and ligating SD after lox511. This vector has a
promoter added thereto. This promoter is not particularly limited.
Any promoter may be used. For example, bacteria- or yeast-derived
promoters described later in the section of transformant
preparation; RNA polymerase promoters such as SP6 RNA polymerase
promoter, T7RNA polymerase promoter, T3RNA polymerase promoter; or
mammal-derived promoters such as EF1 (elongation factor 1)
promoter, PGK (glycerophosphate kinase) promoter, MC1 (polyoma
enhancer/herpes simplex thymidine kinase) promoter may be
enumerated.
2. Gene Trapping
[0087] Two-step gene trapping is performed using the vector
prepared as described above.
[0088] The first step is conventional gene trapping. "Conventional
gene trapping" means to introduce the above trap vector into ES
cells and to trap an endogenous gene present inherently in the ES
cells. By these procedures, the endogenous gene in the ES cells is
disrupted. Using these ES cells, the knockout mice described later
can be prepared. After isolation of the trapped endogenous gene
(FIG. 8; "gene X"), subtle mutations are introduced into this gene
in E. coli using site-specific mutagenesis or the like (FIG. 8;
"gene X"). For the introduction of mutations into gene X, known
techniques such as Kunkel method, gapped duplex method, etc. and
methods based on these techniques may be used. For example,
mutations are introduced by using a mutagenesis kit utilizing
site-specific mutagenesis (e.g. Mutant-K or Mutant-G available from
Takara Shuzo) or LA PCR in vitro Mutagenesis series kit (Takara
Shuzo).
[0089] The second step gene trapping means to introduce into ES
cells the mutated endogenous gene (gene X') ligated downstream of
lox66. By these procedures, recombination occurs between the lox71
site of the trap vector introduced in the first step and the lox66
site of the vector introduced in the second step. As a result, the
modified gene can be introduced into ES cells in the form of a
cassette composed of [(lox71/66)-(gene X')-(loxP)] (FIG. 8).
[0090] According to these procedures, not only modified endogenous
genes but also human genes may be introduced. Any gene may be
introduced. In the present invention, this method is designated
exchangeable gene trapping.
3. Screening for Trap Vector-Integrated Clones (ES Cells)
[0091] If a gene trap vector was introduced into ES cells and then
neomycin resistant clones have been selected from the resultant
cells, these clones are considered to have the trap vector
integrated downstream of a mouse endogenous gene. DNA is extracted
from these clones and analyzed by Southern blotting, to thereby
select clones in which a single copy of the trap vector is
integrated. The inventors have found that this selection method
enables efficient selection of mouse gene-trapping clones.
Therefore, this will be used as a screening system in the present
invention.
(l) Isolation of Neomycin Resistant Clones
[0092] In the present invention, electroporation, microinjection or
the like is used for introducing trap vectors into ES cells. For
example, 100 .mu.g of trap vector is introduced into 3.times.107
TT2 ES cells suspended in 0.8 ml of phosphate buffer by
electroporation (using a BioRad GenePulser at 800 V and 3 .mu.F),
and the resultant cells are cultured in the presence of G418
(concentration: 200 .mu.g/ml). After 1 week, neomycin resistant
clones are isolated.
[0093] The gene trap vector is integrated into the ES cell gnome at
random. Therefore, mere introduction of the trap vector into ES
cells does not necessarily mean integration into a gene. The vector
may be integrated into a non-gene region. However, since the trap
vector contains a drug resistance gene neo (neomycin resistance
gene), those cells expressing this gene are neomycin (also called
G418) resistant. In other words, those cells that survive in the
presence of neomycin are expressing neomycin resistance gene. The
neomycin resistance gene in the trap vector is expressed only when
integrated downstream of a mouse gene which is being expressed in
the ES cells. Thus, the expression of this neomycin resistance gene
means that it has been integrated downstream of a certain gene.
(2) Selection of ES Clones by Integration Pattern
[0094] DNA is extracted from ES clones by conventional methods, and
integration patterns are analyzed by Southern blotting or the like.
When the Southern blot pattern appears as a single band, it can be
judged that only one copy of the vector is integrated. Therefore,
the DNA expressing that pattern is selected. These procedures are
performed in order to select those clones in which isolation of
mouse endogenous genes by plasmid rescue will be easy. Also, those
clones which have become neomycin resistant with only one copy of
the vector are trapping mouse endogenous genes at an extremely high
probability.
4. Establishment of Trap Lines (Transgenic Animals) by Production
Of Chimeric Animals
[0095] Chimeric animals are produced by standard methods (FIG. 9).
The species of chimeric animals produced in the present invention
is not particularly limited. For example, mouse, rat, guinea pig,
rabbit, goat, sheep, pig, dog or the like may be enumerated. In the
present invention, mouse is preferable because of its easy handling
and propagation.
[0096] ES cells selected with neomycin are aggregated with animal
derived-morulae (i.e. aggregates of ES cells and morulae are
formed) to prepare chimeric animal embryos (e.g. those developed to
blastocysts). The resultant embryo is transferred into the uterus
of a foster female animal that has been brought into a
pseudo-pregnant state by mating with a sterile male animal. If the
animal is mouse, offspring will be born about 17 days after this
transfer. Chimeric animals are selected from the offspring animals.
Although those that have a high contribution of chimerism are
likely to be germline chimeric animals, this can be confirmed by
crossing such chimeric animals with normal animals.
[0097] Subsequently, chimeric animals are crossed with normal
female animals to obtain F1 to thereby establish mutant animal
lines. The following analysis is conducted only for those animals
that have been established as trap lines (transgenic animals).
Further, spermatozoa from F1 and two-cell stage embryos obtained by
in vitro fertilization using the spermatozoa can be stored frozen
by ultra-quick freezing technique.
(1) Analysis of Expression Patterns
[0098] F1 animals are crossed and then expression patterns in
embryos (in the case of mouse, 9.5-day embryos) and adult animals
are analyzed.
(2) Analysis of Phenotypes
[0099] For each of the established animal lines, phenotypes of
heterozygous and homozygous animals are analyzed. This analysis is
carried out by macroscopic observation, internal observation by
anatomy, microscopic examination of tissue sections from each
organs, examination of the skeletal system by X-ray photography,
examination of behavior and memory, and blood examination.
(3) Isolation and Structural Analysis of the Trapped Gene and
Preparation of Chromosome Map
[0100] Trapped DNA is isolated from the trap clone, and the
nucleotide sequence thereof is determined (as described later).
Then, homology search is performed using the sequence information
obtained. Consequently, the sequence of the trapped DNA is
classified into one of the groups of known genes, ESTs (expressed
sequence tags), unknown genes or repeats. If the DNA is an EST or
unknown gene, a chromosome map can be prepared. Chromosome maps may
be prepared by fluorescent in situ hybridization (FISH),
association analysis using microsatelite probes or the like, or
analysis of hybrid cells by irradiation. Once the position of the
DNA on the chromosome has been determined, this position is
compared with the positions of mutant genes in existing mutant mice
to examine if the relevant position coincides with one of them.
(4) Construction of Database
[0101] For each of the established lines, database is prepared on
expression patterns of the marker gene in embryos (in the case of
mouse, 10-day embryos) and adult animals; phenotypes in F1 and F2
animals; the nucleotide sequence of the trapped endogenous DNA and,
if the DNA is an EST or unknown gene, its position in the
chromosome.
5. Knockout Animals The knockout animal of the invention is an
animal that has been treated so that the function of a specific
gene is lost. The procedures of such treatment will be described
below.
[0102] The animals that may be used in the present invention
include mouse, rat, guinea pig, rabbit, goat, sheep, pig and dog.
Preferably, mouse is used in the invention because of its easy
handling and propagation.
[0103] A genomic DNA fragment containing an unknown gene is
obtained by PCR from a genomic DNA prepared from animal ES cells or
obtained from a genomic library. Then, this fragment is integrated
into the trap vector of the invention. As a result of this
operation, the function of the exons in the unknown gene is
destroyed. In the trap vector, thymidine kinase (tk) gene or
diphtheria toxin (DT) gene has been ligated in advance for negative
selection. This trap vector is introduced into ES cells by
electroporation or the like. The resultant cells are cultured in
the presence of neomycin for positive selection and a nucleic acid
analogue FIAU (fluoroiodoadenosyluracil) or diphtheria toxin for
negative selection. Through these selections, only the trap
vector-integrated ES cells remain. In these ES clones, the genes
containing disrupted exons are knocked out. The resultant cells are
transferred into the uterus of foster female animals. Then,
chimeric animals are selected from offspring animals. By crossing
these chimeric animals with normal animals, heterozygous animals
are obtained. Then, homozygotes can be obtained through crossing
between the heterozygotes.
[0104] In order to confirm that a knockout mouse is obtained, F1
mice are X-ray photographed and examined for bone abnormalities
(e.g. changes in shape). Alternatively, this confirmation may be
made by observing the presence or absence of abnormalities in the
appearance of mice and by observing abnormalities in various
tissues and organs at the time of anatomy. Also, the confirmation
may be made by extracting RNA from tissues and analyzing the
expression pattern of the relevant gene by Northern blotting. If
necessary, blood samples may be taken and subjected to blood
examination and serum biochemical examination.
6. Isolation of Genes, Construction of Recombinant Vectors and
Preparation of Transformants
(1) Isolation of Genes
[0105] In the present invention, genes trapped as described above
can be cloned and structurally analyzed.
[0106] Isolation of DNA from the trap clone may be performed by
conventional techniques. For example, if cloning is performed using
mRNA from the trap clone, first, total RNA is obtained from the
trap clone by treating the clone with guanidine reagent, phenol
reagent or the like. From the total RNA, poly(A+) RNA (mRNA) is
obtained by affinity column method using, for example, oligo
dT-cellulose or poly U-Sepharose containing Sepharose 2B as a
carrier, or by batch method. Using this mRNA as a template,
single-stranded cDNA is synthesized using oligo dT primers and a
reverse transcriptase. Then, double-stranded cDNA is synthesized
from the single-stranded cDNA. The thus obtained double-stranded
cDNA is inserted into an appropriate expression vector (e.g.
.lamda.gt11) to thereby obtain a cDNA library.
[0107] The gene obtained as described above is subjected to
sequencing. The sequencing may be performed by known techniques
such as the chemical modification method of Maxam Gilbert or the
dideoxy nucleotide chain termination method using DNA polymerase.
Usually, the nucleotide sequence of the gene can be determined
using an automated sequencer. When the 5' region or 3' region of
the relevant cDNA is undetermined, the entire nucleotide sequence
is determined by 5'-RACE or 3'-RACE. RACE (Rapid Amplification of
cDNA Ends) is a well-known technique in the art (Frohman, M. A. et
al., Methods Enzymol. Vol. 218, pp. 340-358 (1993)), and kits for
performing RACE are commercially available (e.g. Marathon.TM. cDNA
Amplification Kit; Clontech). Once the nucleotide sequence of the
gene of the invention has been determined, the gene can be obtained
by chemical synthesis or PCR using primers synthesized based on
that sequence.
(2) Construction of Recombinant Vectors
[0108] A gene fragment of interest is purified and ligated to
vector DNA. As the vector, any vector may be used such phage vector
or plasmid vector. The technique to ligate DNA of interest to
vectors is well known in the art (J. Sambrook et al., Molecular
Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor
Laboratory Press, 1989). Further, recombinant vectors are prepared
from the resultant vector and introduced into E. coli or the like.
Then, appropriate colonies are selected to prepare desired
recombinant vectors.
(3) Transformants
[0109] The transformant of the invention can be obtained by
introducing the recombinant vector of the invention into a host in
such a manner that the gene of interest can be expressed. The host
is not particularly limited as long as it can express the DNA of
the invention. Specific examples of the host include bacteria,
yeast, animal cells and insect cells.
[0110] When a bacterium such as E. coli is used as the host, it is
preferred that the recombinant vector of the invention be capable
of autonomous replication in the bacterium and yet be composed of a
promoter, a ribosome binding sequence, the gene of the invention,
and a transcription termination sequence. A gene that controls the
promoter may also be included. Specific examples of E. coli include
Escherichia coli K12 and DH1, and specific examples of Bacillus
include Bacillus subtilis. As the promoter, any promoter may be
used as long as it can direct the expression of the gene of
interest in the host such as E. coli. For example, E. coli- or
phage-derived promoters such as trp promoter, lac promoter, PL
promoter and PR promoter may be used. An artificially designed and
modified promoter such as tac promoter may also be used. As a
method for introducing the recombinant vector into a host
bacterium, any method for introducing DNA into bacteria may be
used. For example, the method using calcium ions (Cohen, S. N. et
al., Proc. Natl. Acad. Sci. USA, 69:2110-2114 (1972)),
electroporation (Becker, D. M. et al., Methods. Enzymol.,
194:182-187 (1990) or the like may be used.
[0111] When yeast is used as the host, Saccharomyces cerevisiae,
Shizosaccharomyces pombe or the like may be used. As the promoter,
any promoter may be used as long as it can direct the expression of
the gene of interest in yeast. For example, gall promoter, gal10
promoter, heat shock protein promoter, MF .alpha. 1 promoter, PH05
promoter, PGK promoter, GAP promoter, ADH promoter and AOX1
promoter may be enumerated. As a method for introducing the
recombinant vector into yeast, any method for introducing DNA into
yeast may be used. For example, electroporation, the spheroplast
method (Hinnen, A. et al., Proc. Natl. Acad. Sci. USA, 75:1929-1933
(1978), the lithium acetate method (Itoh, H., J. Bacteriol.,
153:163-168 (1983) or the like may be used.
[0112] When an animal cell is used as the host, COS cells, Vero
cells, Chinese hamster ovary cells (CHO cells), mouse myeloma cells
or the like may be used. As the promoter, SR.alpha. promoter, SV40
promoter, LTR promoter, EF1 promoter, PGK promoter, MC1 promoter,
or the like may be used. Alternatively, human cytomegalovirus early
gene promoter or the like may be used. As a method for introducing
the recombinant vector into an animal cell, electroporation, the
calcium phosphate method, lipofection, or the like may be used.
[0113] When an insect cell is used as the vector, Sf9 cells, Sf21
cells or the like may be used. As a method for introducing the
recombinant vector into an insect cell, the calcium phosphate
method, lipofection, electroporation, or the like may be used.
BRIEF DESCRIPTION OF THE DRAWINGS
[0114] FIG. 1 is a diagram showing the concept of structural
analysis in both gene region and non-gene region.
[0115] FIG. 2 is a diagram showing an outline of the construction
of a trap vector and the gene trapping according to the
invention.
[0116] FIG. 3 is a diagram showing the structure of loxP (top
sequence-SEQ ID NO: 3; bottom sequence-SEQ ID NO: 17).
[0117] FIG. 4 is a diagram showing recombination between lox71 and
lox66 (SEQ ID NOS: 15 (lox71), 16 (lox66), 6 (lox71/66), and 3
(loxP)).
[0118] FIG. 5 is a diagram showing insertion of a DNA fragment when
mutant loxP sequences are used.
[0119] FIG. 6 is a diagram showing trap vectors of the
invention.
[0120] FIG. 7 is a flow chart showing the construction of a trap
vector pU-Hachi.
[0121] FIG. 8 is a diagram showing the two-step exchangeable gene
trapping of the invention.
[0122] FIG. 9 is a diagram showing an outline of the establishment
of trap lines by production of chimeric animals.
[0123] FIG. 10 is a diagram showing various positions of
integration of trap vectors.
[0124] FIG. 11 is a photograph showing a bending mutation in the
tailbone in mouse.
BEST MODES FOR CARRYING OUT THE INVENTION
[0125] Hereinbelow, the present invention will be described in more
detail with reference to the following Examples. It should be noted
that the technical scope of the present invention is not limited by
these Examples.
EXAMPLE 1
Construction of Varied-Type Gene Trap Vectors
[0126] (1) Construction of pU-Hachi Trap Vector
[0127] pU-Hachi vector is derived from pGT1.8IRES .beta.-geo, and
contains SA sequence from mouse En-2 gene and .beta.-geo sequence
linked to encephalomyocarditis virus-derived IRES sequence. First,
a BamHI fragment of lox71 is inserted into the BglII site of
pGT1.8RES .beta.-geo. Then, plasmid pEBN-SE7ti was constructed by
inserting a 180 bp (SP) sequence (which is a part of rabbit .beta.
globin gene), loxP sequence, and poly A addition signal from mouse
phosphoglycerate kinase-1 (PGK) into a modified vector pUC19 from
which lacZ sequence has been removed. The SP sequence was used to
protect the 3' end of the trap vector. By inserting a SalI fragment
of SA-IRES-lox71-.beta.-geo into the SalI site of pEBN-SE7ti,
pU-Hachi was obtained.
(2) Construction of pU-12 Trap Vector
[0128] In order to construct pU-12 trap vector, first, the PGK
poly(A) signal of pE3NSE7 was replaced with puromycin resistance
gene+PGK poly(A) signal. Then, lox511 was inserted into the BglII
site downstream thereof to prepare a plasmid. By inserting a SalI
fragment of SA-IRES-lox71-.beta.-geo from pU-Hachi into the SalI
site of the above plasmid, pU-12 was obtained.
(3) Construction of pU-17 Trap Vector
[0129] First, lox511, loxP, PGK poly(A) signal and lox2272 were
inserted in this order into plasmid pSP73 (Promega) to construct
pSP5PP2. Subsequently, pU-Hachi was cleaved at one of the two BamHI
sites within SA located upstream of the other. The DNA fragment of
pU-Hachi up to the upstream BamHI site in SA, lox71 sequence, the
DNA fragment of pU-Hachi from the downstream BamHI site in SA to
the KpnI site, and a NcoI-SalI fragment of .beta.-geo were inserted
in this order into pBluescriptII KS+plasmid to construct pKS+S71A
.beta.-geo. From this plasmid, an XbaI fragment of SA-.beta.-geo
containing lox71 was excised and inserted into the SpeI site of
pSP5PP2 to thereby obtain pU-17.
EXAMPLE 2
Selection of ES Cell Clones
[0130] In the electroporation using pU-Hachi trap vector, 100 .mu.g
of SpeI-digested DNA and 3.times.10.sup.7 cells were used. Cells
were suspended in 0.8 ml of PBS and electroporated using a BioRad
GenePulser at 800 V and 3 .mu.F. After 48 hours, the cells were
cultured in the presence of 200 .mu.g/ml G418. This selection was
maintained for 7 days. The resultant colonies were plated on
24-well plates for propagation and stored frozen. Trap clones were
analyzed by Southern blotting to select those cell strains that
exhibit patterns of single copy integration.
[0131] In order to remove .beta.-geo sequence from the trap clone,
pCAGGS-Cre (Araki, K. et al., Proc. Natl. Acad. Sci. USA,
92:160-164, 1995; Araki, K. et al., Nucl. Acids Res., 25:868-872,
1997; Araki, K. et al., J. Biochem. Tokyo, 122: 977-982, 1997) was
electroporated in a circular form. This electroporation was carried
out under the same conditions as described above except that the
number of cells was 1.5.times.10.sup.7 and that the PBS volume was
0.4 ml.
[0132] One half of the thus treated cells were plated onto a 100 mm
plate and grown for 48 hours. Then, the cells were re-plated onto
100 mm plates at 1.times.10.sup.3 cells per plate for colony
formation. After 1 week, colonies were picked up and expanded for
DNA preparation.
[0133] For the co-electroporation experiments designed for targeted
integration into the lox71 site of the trap vector, 20 .mu.g of
each targeting plasmid (p66.sup.2IEGPPac, p66.sup.2INZPPac or
p66PGKPac-5) and pCAGGS-Cre were used in circular forms.
[0134] Plasmid p66PGKPac-5 was constructed by inserting a lox66
fragment and PGK promoter-puromycin resistance gene coding sequence
into pSP73 vector (Promega). Plasmid p66.sup.2IEGPPac was
constructed from pSP73 vector (Promega), IRES sequence, EGFP gene
(Clontech), PGK promoter, Pca gene and lox66 sequence. Plasmid
p66.sup.2INZPPac was constructed by replacing the PGK gene in
p66.sup.2IEGPPac with a lacZ gene fused to SV40 large T
gene-derived nuclear localization signal.
[0135] Cells suspended in PBS (1.times.10.sup.7 cells/0.8 ml) were
electroporated at 200 V and 950 .mu.F. After 48 hours, the cells
were subjected to selection with puromycin at 2 .mu.g/ml for 3
days. Then, the cells were transferred into a normal medium. Nine
days after the electroporation, colonies were picked up and
expanded.
[0136] Embryoid bodies (EBs) were produced according to a known
method (Abe, K., Niwa, H. et al., Exp. Cell Res. 229: 27-34, 1996).
.beta.-galactosidase activity in ES cells and EBs was determined by
staining with 5-bromo-4-chloro-3-indolyl .beta.-D-galactopyranoside
(X-gal) as described (Glossler, A. and Zachgo, J., Gene Targeting:
A Practical Approach, Joyner , A. (ed.), Oxford University Press,
Oxford, 1993, pp. 181-227).
[0137] Trap vector pU-Hachi was linearized and introduced into TT2
ES cells. As a result, 109 clones were isolated. Genomic DNA was
prepared from each clone and subjected to Southern blotting using a
pUC probe and at least 3 restricting enzymes to examine trap
vector-integration patterns.
[0138] A single band was confirmed in 69% of the clones. Since the
presence of lox71 site is essential for the Cre-mediated
integration, the presence was confirmed by Southern blotting using
a lacZ probe and PstI digestion. As a result, it was found that
lox71 was deleted in 10% of the clones (Table 4). The remaining 59%
of the clones in which a single copy was integrated and yet lox71
site was maintained were selected for further analysis.
TABLE-US-00004 TABLE 4 Single copy integration (%) Multi-copy Total
No. Clones retaining lox71 site integration (%) of clones Retaining
plasmid Without plasmid Clones without 2-3 .gtoreq.5 tested (%)
replication origin replication origin lox71 site copies copies 109
(100) 24 (22) 40 (37) 11 (10) 26 (24) 8 (7)
[0139] In order to evaluate the capture of endogenous genes by the
trap vector, cells were stained with X-gal before and after the
formation of embryoid bodies. As shown in Table 5, 97% of the
tested clones exhibited .beta.-gal activity at a specific stage in
differentiation. This means that pU-Hachi trap vector performs
effective gene trapping comparable to the trapping of other
IRES-.beta.-geo vectors.
TABLE-US-00005 TABLE 5 Expression of .beta.-geo Clone No. (%)
Undifferentiated ES Cells Differentiated EBs (Day 8) 26(41) + +
32(50) - + 4(6) + - 2(3) - -
EXAMPLE 3
Selection Frequency of Clones
[0140] In order to select those clones in which a single copy of
the trap vector was integrated, DNA was extracted from the
selected, neomycin resistant clones and analyzed by Southern
blotting.
[0141] Briefly, cells were lysed with SDS/proteinase K, treated
with phenol/chloroform (1:1, vol:vol) twice, precipitated with
ethanol, and then dissolved in TE buffer (10 mM Tris-HCl, pH 7.5/1
mM EDTA). Six micrograms of genomic DNA was digested with
appropriate restriction enzymes, electrophoresed on 0.9% agarose
gel and then blotted onto a nylon membrane (Boehringer Mannheim).
Hybridization was performed using a DIG DNA Labeling and Detection
Kit (Boehringer Mannheim).
[0142] For PCR analysis, DNA was subjected to 28 cycles of
denaturation at 94.degree. C. for 1 min, annealing at 55.degree. C.
for 2 min and extension at 72.degree. C. for 2 min in the reaction
solution described below.
[0143] The primers used in the PCR were as follows:
.beta.-geo detection primers
TABLE-US-00006 Z1 (forward): 5'-gcgttacccaacttaatcg-3' (SEQ ID NO:
7) Z2 (reverse): 5'-tgtgagcgagtaacaacc-3' (SEQ ID NO: 8)
Primers for detecting the replication origin region in pUC
vector
TABLE-US-00007 Ori2 (forward): 5'-gccagtggcgataagtcgtgtc-3' (SEQ ID
NO: 9) Ori3 (reverse): 5'-cacagaatcaggggataacgc-3' (SEQ ID NO:
10)
TABLE-US-00008 Reaction Solution 10 x PCR buffer 2 .mu.l 10 mM dNTP
0.2 .mu.l Forward primer (100 pmol/.mu.l) 0.2 .mu.l Reverse primer
(100 pmol/.mu.l) 0.2 .mu.l AmpliTaq DNA polymerase (Perkin Elmer)
0.2 .mu.l Total Volume (adjusted with sterilized distilled water)
20 .mu.l
[0144] One half of the resultant PCR product was loaded onto
agarose gel and analyzed.
[0145] Plasmid rescue (i.e. recovery of the trapped gene) was
performed as described below.
[0146] Briefly, genomic DNA (20 .mu.g) was digested with
appropriate restriction enzymes and ligated in a reaction volume of
400 .mu.l to obtain circular molecules. After phenol/chloroform
extraction and ethanol precipitation, the DNA was suspended in 10
.mu.l of TE. Using one half of this DNA suspension, E. coli (STBL2;
Life Technologies) was transformed by electroporation. The
electroporation was performed according to the manual of BioRad
GenePulser. The electroporated cells were incubated in 1 ml of
Circle Grow medium (BIO 101) at 30.degree. C. for 1 hour under
agitation. Then, after concentration, the sample was plated on
LB/agar plates followed by selection of plasmids with ampicillin.
The rescued plasmids were analyzed by restriction mapping and
sequencing. Nucleotide sequences were determined with Thermo
Sequenase Fluorescent-Labeled Primer Cycle Sequencing Kit
(Amersham).
[0147] As a result, as shown in Table 6, clones in which
recombination occurred at a high frequency could be obtained.
TABLE-US-00009 TABLE 6 Length of the 5' No. of subclones flanking
region Length of the 3' No. of in which obtained by flanking region
subclones recombination Recombination plasmid rescue obtained by
plasmid Trap Clone analyzed occurred frequency (%) (kb) rescue (kb)
Ayu8-003 23 15 65 75 53 Ayu8-016 20 2 10 3.8 4.5 Ayu8-025 23 16 70
1.8 6.5 Ayu8-104 12 5 42 3.5 7 Ayu8-108 12 6 50 5 6
EXAMPLE 4
Production of Chimeric Mice and Gene Analysis
[0148] (1) Introduction of the Clone into Mice
[0149] The trap ES clone was aggregated with ICR mouse-derived 8
cell stage embryos and cultured overnight. On the next day,
aggregates of the ES cell and embryo that had developed to
blastocysts were selected. Approximately 20 of these chimeric
embryos were transferred into the uterus of a foster female mouse
that was pre-mated with a sterile male mouse. Offspring mice were
born about 17 days thereafter. Eight weeks after birth when they
became sexually mature, these chimeric mice were crossed with
normal female mice to obtain ES clone-derived F1 mice.
(2) Analysis of Phenotypes
[0150] The F1 mice were X-ray photographed, and the presence of
absence of abnormalities in the bone was observed.
(3) Analysis of the Trapped Gene
[0151] Since the trapped gene must be forming a fusion mRNA with
.beta.-geo, the trapped gene was identified utilizing this
presumption.
[0152] Briefly, mRNA was extracted from X-gal staining-positive
tissues of F1 mice. From the resultant mRNA, single-stranded cDNA
was synthesized with a Thermoscript RT-PCR system (GIBCO BRL) using
sequences within the SA as primers. Subsequently, a cDNA fragment
corresponding to the upstream region of the trapped gene that was
linked to the exon of the SA in the vector was obtained using a
5'RACE system (GIBCO BRL). The resultant cDNA fragment was cloned
into a plasmid vector and subjected to sequencing.
(4) Results
[0153] Table 7 shows one example of the results obtained from the
analysis of trapped genes.
TABLE-US-00010 TABLE 7 Clone No. Gene Phenotype 1 Ayu8-R38 Sp1 2
Ayu8-029 PCM1 (pericentriol material 1) 3 Ayu3-008 Cyclin B2 4
Ayu6-003 Homologue to the E. coli Ftsj1 gene 5 Ayu8-003 dynamin II
Death at embryonic stage 6 Ayu8-R16 sui1 7 Ayu8-016 Upstream region
of hnRNP L 8 Ayu8-019 RNA polymerase I 9 Ayu8-108 importin .beta.
10 Ayu8-021 Unknown Kinky tail
[0154] Among the genes obtained as described above, PCM1 gene was
analyzed. As a result, sequences shown in SEQ ID NOS: 11 to 13
(5'RACE partial fragments) were obtained. These sequences matched
with a part of the known PCM1 gene. Further, the mice obtained from
Ayu8-021 clone exhibited a mutation of bending in the tail bone
(kinky tail) (FIG. 11). This mutant gene fragment was sequenced to
thereby obtain the sequence as shown in SEQ ID NO: 14.
[0155] All of the publications, patents and patent applications
referred to in the present specification are incorporated herein by
reference in their entirety.
INDUSTRIAL APPLICABILITY
[0156] The present invention provides gene trap vectors and a
method of gene trapping. According to the present invention, first,
(1) knockout mice can be produced efficiently. In most cases, mouse
genes are knocked out by the integration of the trap vector into
genes. Therefore, if the trap vector-introduced ES cells are used,
a mouse gene-knocked out mice can be produced. That is, knockout
mice can be produced efficiently by selection of neomycin resistant
clones and selection of those clones in which a single copy of the
trap vector is integrated. According to conventional homologous
recombination methods, one researcher can produce only 4 lines of
knockout mice in one year at his/her best. According to the method
of the invention, however, one researcher can establish as many as
240 lines in one year if, for example, he/she establishes 6 lines
per week and works 40 weeks a year. Thus, the method of the
invention is 60 times more efficient than conventional methods.
[0157] (2) The method of the invention allows detailed analysis of
gene functions.
[0158] In the method of gene trapping of the invention, it is
possible to introduce mutations in advance into each part of a gene
that seems to have a certain function, and integrate the resultant
mutant genes into trap vectors. Then, the mutant gene-integrated
trap vector can be introduced into mouse followed by analysis of
the phenotype.
[0159] (3) The method of the invention allows the production of
disease model mice which are closer to human.
[0160] According to the present invention, it is possible to create
disease model mice which are closer to human than conventional
models because a human gene having the same mutation as found in a
human disease can be introduced into mice replacing the
corresponding mouse gene.
SEQUENCE LISTING FREE TEXT
[0161] SEQ ID NO: 1: synthetic DNA SEQ ID NO: 2: synthetic DNA SEQ
ID NO: 3: synthetic DNA SEQ ID NO: 4: synthetic DNA SEQ ID NO: 5:
synthetic DNA SEQ ID NO: 6: homologous recombination sequence SEQ
ID NO: 7: synthetic DNA SEQ ID NO: 8: synthetic DNA SEQ ID NO: 9:
synthetic DNA SEQ ID NO: 10: synthetic DNA
REFERENCES
(1) Relating to Gene Trapping:
[0162] 1) Wurst, W. et al., Genetics 139: 889-899, 1995.
[0163] 2) Chowdhury, K. et al., Nucleic Acids Res. 25:1531-1536,
1997.
[0164] 3) Hicks, G. G. et al., Nature Genetics 16: 338-344,
1997.
[0165] 4) Zambrowicz, B. P. et al., Nature 392: 608-611, 1998.
(2) Relating to the Cre-loxP System
[0166] 1) Sauer, B. and Henderson, N. Proc. Natl. Acad. Sci. USA
85: 5166-5170, 1988.
[0167] 2) Lakso, M. et al., Proc. Natl. Acad. Sci. USA 89:
6232-6236, 1992,
[0168] 3) Gu, H. et al., Independent control of immunoglobulin
switch recombination at individual switch regions evidenced
1993.
[0169] 4) Albert, H. et al., Plant J. 7: 649-659, 1995.
[0170] 5) Schwenk, F. et al., Nucleic Acids Res. 23: 5080-5081,
1995.
(3) List of References relating to Gene Trapping
[0171] 1) Miyazaki, J. et al., Gene 79: 269-277, 1989.
[0172] 2) Niwa, H. et al., Gene 108: 193-200, 1991.
[0173] 3) Niwa, H. et al., J. Biochem, 113: 343-349, 1993.
[0174] 4) Niwa, H. et al., Gene 169: 197-201, 1996.
[0175] 5) Abe, K., Niwa, H. et al., Exp. Cell Res. 229: 27-34,
1996.
[0176] 6) Araki, K. et al., Nucleic Acid Res. 25: 868-872,
1997.
[0177] 7) Araki, K. et al., J. Biochem. 122: 977-982, 1997.
[0178] 8) Oike, Y. et al., Human Mol. Genet-In Press
[0179] 9) Oike, Y. et al., Blood in press
Sequence CWU 1
1
17113DNAArtificial SequenceDescription of Artificial
Sequencesynthetic DNA 1taccgttcgt ata 13213DNAArtificial
SequenceDescription of Artificial Sequencesynthetic DNA 2tatacgaacg
gta 13334DNAArtificial SequenceDescription of Artificial
Sequencesynthetic DNA 3ataacttcgt atagcataca ttatacgaag ttat
34413DNAArtificial SequenceDescription of Artificial
Sequencesynthetic DNA 4ataacttcgt ata 13513DNAArtificial
SequenceDescription of Artificial Sequencesynthetic DNA 5tatacgaagt
tat 13634DNAArtificial SequenceHomologous recombination sequence
6taccgttcgt atagcataca ttatacgaac ggta 34719DNAArtificial
SequenceZ1 Forward primer used in PCR for B-geo detection
7gcgttaccca acttaatcg 19818DNAArtificial SequenceZ2 reverse primer
used in PCR for B-geo detection 8tgtgagcgag taacaacc
18922DNAArtificial SequenceOri2 forward primer used in PCR for
detecting the replication origin region in pUC vector 9gccagtggcg
ataagtcgtg tc 221021DNAArtificial SequenceOri3 reverse primer used
in PCR for detecting the replication origin region in pUC vector
10cacagaatca ggggataacg c 2111400DNAMus
musculusmisc_feature(36)..(36)n is a, c, g or t 11agaaacttaa
acagcggata aacttcagtg atttanatca gagaagtatt ggaagtgatt 60ctcaaggtan
agcaacagcg gctaacaaca aacgtcagct tagtgaaaac cgaaagccct
120tcaacttttt gcctatgcag attaatacta acaagagcaa ggatgctact
gcaagtcttc 180caaagagaga gatgacaacg tcagcacagt gcaaagagtt
gtttgcttct gctctaagta 240atgacctttt gcaaaactgt caatctctga
agaagatggg agaggggagc ctgcatggga 300aacaccagat tgtaagcagg
cttgttcaat cctgactata ttactaaagc tagttctatg 360cnanaagttt
tgtaaanaaa atgaaagtct gcaatgttga 40012416DNAMus
musculusmisc_feature(37)..(37)n is a, c, g or t 12tcttctagct
ttgcagcata aagcagagca agctatnagc tgtgatggat gactctgttg 60ttacagaaac
tacaggaagc ttatctggag tcagcatcac atctgaacta aatgaagaac
120tgaatgattt aattcagcgt ttccataatc agcttcgtga ttctcagcct
ccagctgttc 180cagacaacag aagacaggca gaaagtcttt cattaactag
agagatttct cagagcagaa 240atccctcagt ttctgaacat ttacctgatg
agaaagtaca gctttttagc aaaatgagag 300tactacagga aaagaacaag
aaatggacaa attagttggg agaacttcat aaccttcgag 360atnagcatct
gaacaactca tcatttgtgc cntcaacttc ncnccaaaga agtggg 41613484DNAMus
musculusmisc_feature(33)..(33)n is a, c, g or t 13gtttctacac
ctactgaaca gcagcagcca ttnagctcaa aatccttnca gggnaaaaca 60gagtatatgg
cttttccaaa accctctgna aagcagttct tctcttggag cagaaaagca
120aaggaatcaa gaaacagccc gaagaggaag ctgaaaacac taagacacca
tggttatatg 180atcaagaagg tggagtagaa aaaccatttt tcaagactgg
atttacagag tctgtagaga 240aagntacaaa atagtanccg caaaaatcaa
ccagatacaa gcaggagaag acgtcggttt 300gatgaagaat cccttggaaa
gctttagcag tatgcctgat cctatagacc caacatcagt 360aactaaaaca
tttaaaacaa gaaaagcatc tgcccaggcc agcctggcct ctaaggacaa
420aactcccaaa tcaaagagta agaagaggat tctactcagc tgaaaagtag
agttaaaaat 480attg 48414211DNAMus musculus 14ctgtctgtca ttgtcgttct
cctttagaag gcagaaaaga aatgggaaga aaaaaggcaa 60aatctggaac actataacgg
aaaggagttc gagaagctcc tggaggaagc tcaggccaac 120atcatgaagt
caattccaaa cctggagatg cccccagctt ccagcccagt gtcaaaggga
180gatgcggcag gggataagct ggagctgtca g 2111534DNAArtificial
SequenceDescription of Artificial Sequencesynthetic DNA
15taccgttcgt atagcataca ttatacgaag ttat 341634DNAArtificial
SequenceDescription of Artificial Sequencesynthetic DNA
16ataacttcgt atagcataca ttatacgaac ggta 341734DNAArtificial
SequenceDescription of Artificial Sequencesynthetic DNA
17tattgaagca tatcgtatgt aatatgcttc aata 34
* * * * *