JP2002233374A - Vector for creating non-human animal in which gene is transduced and/or deleted - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a vector, for creating a transgenic non-human animal, having a function that permits site-specifically transducing only one copy of objective gene such as lethal gene, a function that permits controlling an expression time of the gene, and a function that permits early detection of an expression site of the gene, and a cell in which at least a part of the DNA of the vector is incorporated, a transgenic non-human animal created using the vector or cell. SOLUTION: This vector is constructed so as to contain a DNA prepared by linking a promoter gene, a DNA that has recombinase-recognizing sequence loxP and encodes fluorescent protein EGFP fused with SV40 poly A signal in both sides, and β-globin poly-A signal having a multi-cloning site for introducing an objective gene in the upstream side in this order.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a gene transfection having both a function of controlling the expression time of a target gene, particularly a lethal gene, and a function of detecting the expression site of the gene at an early stage. And / or a vector for producing a gene-deficient non-human animal, preferably a vector for producing a gene-transferred and / or gene-deficient non-human animal which also has a function of limiting the number of copies of a target gene to one copy and site-specifically introducing it. The present invention also relates to a transgenic or gene-deficient non-human animal obtained by using such a vector.

[0002]

2. Description of the Related Art In recent years, with the development of molecular biological techniques,
Genes have been relatively easily isolated and their structural analysis performed. However, it is difficult to analyze the function of a gene or the pathogenesis of a disease only by isolating and analyzing the structure of the gene. In 1980, a transgenic mouse (transgenic mouse) was first produced by injecting the isolated gene into a mouse fertilized egg, and in 1989, a mouse in which a specific gene was deleted by target gene recombination, Knockout mice were created. These gene transfer and gene deletion technologies are widely used in many fields such as functional analysis of genes at the individual level, development of human disease model animals, breeding as industrial applications, and production of useful substances. I have.

[0003] As a method for preparing the above-mentioned transgenic animal, a method by microinjection of DNA into a pronucleus of a fertilized egg, a method of preparing a chimera after introducing DNA into embryonic stem cells (ES cells), or a method of initializing a retroviral vector Methods for infecting and introducing a developing embryo are known. The transgenic animals obtained by these methods usually have a foreign gene stably integrated into a part of its chromosome. However, it is not possible to integrate the transgene into a desired position on the animal chromosome by conventional methods, so it is necessary to efficiently express the transgene at the individual level or to identify the specificity of its expression function. It was difficult. Recently, bacteriophage P1 C
re-lox recombination system (J. Mol. Biol. 150, 467-48)
6, 1981) and the yeast Saccharomyces cerevische FL
P-FRT recombination system (J. Mol. Biol. 284, 363-38)
4,1998). These are used as tools for regulating the expression time in a tissue-specific or differentiation-specific manner.

[0004] Further, in the conventional method used for introducing a heterologous DNA into an embryonic cell line in the production of a transgenic animal, the site of integration of the foreign gene into the genome and the copy number of the introduced DNA are controlled. Can not do. Integration of the gene of interest occurs randomly, often multiple copies of the gene are integrated simultaneously as double-tailed, and the site of integration and the number of foreign genes integrated generally vary from transgenic animal to animal. As a result, the function of the endogenous gene located at the insertion site is inactivated, and there has been a problem that the deletion of the function of the endogenous gene due to many random insertions cannot be easily detected. When these gene products are important for the development and growth of animals, serious disruptions will occur in the developmental form and growth of animals, and random insertion of the target gene will occur at sites inappropriate for gene expression. Therefore, the insertion site and the number of insertions are expected to be different between animals, and the expression of the gene is not stable, and it has been extremely difficult to analyze the function of the target gene.

[0005] On the other hand, lethal genes that are caused by gene deletion or mutation and that inhibit normal growth of an individual and cause death before the end of their lifespan are caused by a dominant lethal mutation that causes death in a heterologous state. Genes and recessive lethal genes that do not have a lethal effect unless they become homozygous are known, and these lethal genes are isolated and identified year by year,
That number is growing. However, even if a lethal gene such as a toxic gene is introduced into a cell or animal individual for functional analysis of the gene, and the gene product is expressed in a small amount, the gene is randomly integrated on the chromosome. In the case of (1), a large number of cells are introduced as a result, and there is a problem that the cells or the animal individuals themselves are killed by replication at a high copy number. From this, the foreign gene can be
It has been desired to produce transgenic animals by introducing them into cells or animal individuals with a limited copy or low copy number.

[0006]

In order to prepare an appropriate disease model animal by gene transfer, a vector for introducing a target gene is devised so as to avoid lethality of the animal. It is desired to be able to control the expression timing, site and amount of the gene.
Vectors satisfying these conditions are useful for elucidating the mechanism of genetic diseases and for preparing model animals for therapeutic development. An object of the present invention is to introduce and / or delete a gene having both a function of controlling the expression time of a target gene such as a lethal gene and a function of detecting the expression site of the gene at an early stage. A vector for producing a non-human animal, preferably a vector for producing a gene-introduced and / or non-human animal having a gene-deficient gene, which also has a function of introducing a target gene into one copy in a site-specific manner, and using such a vector. And / or a gene-deficient non-human animal or the like produced by the method.

[0007]

Means for Solving the Problems Production of transgenic mouse animals is an essential technique for elucidating molecular functions and developing therapeutic drugs. However, depending on the gene to be introduced, many are lethal to ontogenesis, and moreover,
It is difficult to control the expression of a target gene in a site-specific and time-specific manner. Therefore, the present inventors have a promoter gene, a recombinase recognition sequence at both ends,
DNA encoding a fluorescent protein to which a poly-A signal is linked, and poly-A signal having a multi-cloning site for introducing a target gene on the upstream side are connected in order of D
A vector linked with NA is prepared, and the target gene is incorporated into the multicloning site for introducing the target gene,
Gene transfer into COS7 cells,
Induction of the expression of the target gene was observed by Adeno-cre infection or co-introduction of a Cre expression vector into S7 cells. Further, when a transgenic mouse was prepared by microinjecting the vector into which the target gene was incorporated into a mouse fertilized egg, it was possible to know the organ / organ that expresses the target gene at an early stage. Adeno-cre infection of fetal fibroblasts derived from the transgenic mice induced expression of the target gene. The present invention has been completed based on these findings.

[0008] That is, the present invention relates to a vector for introducing a gene of interest and / or for producing a gene-deficient non-human animal, the function of which can control the expression time of the gene of interest. A vector (Claim 1), which has a function of early detecting the expression site of the target gene,
The vector (claim 2) according to claim 1, which has a function of limiting the target gene to one copy and introducing the gene in a site-specific manner, and a gene-introduced and / or gene-deficient non-human. The animal production vector is ligated in the order of a promoter gene, DNA encoding a fluorescent protein fused with a polyA signal having a recombinase recognition sequence at both ends, and a polyA signal having a target gene introduction multicloning site on the upstream side. The vector according to claim 1 or 2, wherein the recombinase recognition sequence is a loxP or FRT sequence. (Claim 4) Alternatively, the poly-A signal is a β-galactosidase gene, SV4
0, β-globin gene, α-globin gene, or poly A signal derived from human growth hormone gene, the vector according to claim 3 or claim 4 (claim 5), and the fluorescent protein is GFP, EGFP (Enhanc
ed GFP), EYFP (Enhanced Yellow Fluorescent Pr
otein), ECFP (enhanced CYAN fluorescent protei
n) or DsRed.
The present invention relates to the vector according to any one of claims 1 to 5, (claim 6).

[0009] The present invention also provides the vector according to any one of claims 1 to 6, wherein the negative selectable marker gene is a thymidine kinase. The vector according to claim 7 (claim 8), wherein
8. A target gene transfer vector, wherein the target gene has been introduced into the vector for gene transfer and / or the production of a gene-deficient non-human animal according to any one of claims 8 to 10.
10. The target gene transfer vector according to claim 9, wherein the target gene is a lethal gene.
0).

[0010] The present invention also provides a cell (claim 11) into which all or a part of the DNA of the target gene transfer vector according to claim 9 or 10 has been introduced, and a cell according to claim 9 or 10. A method for producing a gene-introduced or gene-deficient non-human animal, which comprises using the DNA of the whole or a part of the target gene-introducing vector and the cell according to claim 11 (claim 12), and the method according to claim 12. The transgenic or genetically deficient non-human animal obtained by the production method (claim 13) or the non-human animal is a mouse, rat, rabbit, pig, goat, or cow. The transgenic or gene-deficient non-human animal according to claim 13 (claim 14), or the claim 13 or 14
A cell, tissue or organ obtained from the transgenic or gene-deficient non-human animal according to claim (claim 15), the cell according to claim 11, the transgenic or gene-deficient non-human animal according to claim 13 or 14, or a claim. Item 15. The cell according to Item 15,
The present invention relates to a cell, tissue, organ or transgenic or gene-deficient non-human animal expressing a target gene obtained by treating a tissue or organ with a recombinase (Claim 16).

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION The vector for introducing a gene and / or producing a gene-deficient non-human animal of the present invention has a function capable of controlling the timing of expression of a target gene such as a lethal gene, and a site where the gene is expressed. The vector is not particularly limited as long as it is a vector for gene introduction and / or gene-deficient non-human animal production which also has a function of early detection of the target gene. A gene transfer and / or a vector for producing a gene-deficient non-human animal, which also has a function that can be introduced into, is preferable, for example, a promoter gene,
D encoding a fluorescent protein having a polyA signal fused downstream having recombinase recognition sequences at both ends
NA, and a DN connected in the order of poly A signal having a multiple cloning site for introducing a target gene on the upstream side.
Specific examples include vectors containing A. FIG. 1 shows an outline of the vector for gene introduction and / or gene-deficient non-human animal production of the present invention.

The function capable of controlling the expression time of the target gene refers to a function capable of regulating the expression of the target gene at a desired time. For example, the function capable of controlling the bacteriophage P1 downstream of the promoter. A polyA signal having a multi-cloning site for introducing a target gene on the upstream side is sequentially linked via a recombinase recognition sequence such as a loxP sequence recognized by Cre recombinase or an FRT sequence recognized by FLP recombinase of yeast Saccharomyces cerevisiae. Can provide the above function. For example, by expressing recombinase cre such as infecting Adeno-cre at a desired time, the target gene can be expressed at a desired time (see FIG. 1). In addition, by using these Cre-loxP system and FLP-FRT system, not only can the expression time of a target gene be controlled, but also tissue-specific analysis and expression analysis of a lethal gene, which has been difficult in the past. Can also be done.

In the present invention, the function of detecting the expression site of the target gene at an early stage means that the vector for introducing the gene of the present invention and / or the gene-deficient non-human animal is introduced into non-human animal cells. In the case of inducing gene expression, it refers to the function of knowing in advance which site to express. For example, GFP (green), EGFP (Enhanced GFP; green), EYFP (Enhanced Yellow Fluorescent) between recombinase recognition sequences. Protei
n; yellow), ECFP (enhanced CYAN fluorescent prot
The above function can be imparted by inserting a fusion gene of a polyA signal and a gene encoding a fluorescent protein such as ein) (blue) or DsRed (red).

In the present invention, the function of introducing a target gene in a site-specific manner limited to one copy means that the target gene is introduced into a specific site on the chromosome of a non-human animal cell, and then Cre expression is performed. Refers to a function in which the transgene copy number is limited to one copy along with, for example, a unique site located at both ends of a series of units in the gene introduction and / or gene-deficient non-human animal production vector of the present invention, for example, SalI / XhoI of the vector shown in FIG.
This can be achieved by incorporating genomic fragments before and after the site where the target gene is to be introduced into the site, and homologously recombining the ES cell or fertilized egg with the site-specific introduction of the target gene. In addition, after performing homologous recombination, a target homologous recombinant (ES clone) can be easily selected with a drug or the like, for example, a thymidine kinase (PGK-tk) gene of phosphoglycerokinase, diphtheria toxin A Fragment (DT-A)
Gene, the herpes simplex virus thymidine kinase (H
It is particularly preferable to introduce a negative selectable marker gene such as the SV-tk) gene into the gene introduction and / or gene-deficient non-human animal production vector of the present invention. Note that these negative selectable marker genes are not usually used in existing transgenic vectors.

The promoter used in the present invention is not particularly limited as long as it can stably express a downstream gene. Examples thereof include a CAG promoter, a β-actin promoter, and a retrovirus LTR.
Promoter, adenovirus major late promoter,
Mouse phosphoglycerokinase promoter, RN
Specific examples thereof include A polymerase II promoter, SV40 early promoter, SV40 late promoter, metallothionein promoter, and herpes simplex virus thymidine kinase promoter. Also, an enhancer can be provided upstream of the promoter.

In the present invention, the poly-A signal in the fusion gene of the gene encoding the fluorescent protein and the poly-A signal includes β-galactosidase gene, SV4
Specific examples of the poly-A signal derived from 0, β-globin gene, α-globin gene, human growth hormone gene and the like can be specifically exemplified, but not limited thereto. In addition, a multiple cloning site for introducing a target gene [Mlu on the upstream side, preferably on the upstream side and downstream side.
I / PstI / HindIII / BstBI / EcoRI
/ SacI / SmaI / XmaI / KpnI / NcoI
/ SphI / NsiI / ClaI / BglII].
For example, β-galactosidase gene, SV40, β-
Specific examples include polyA signals derived from globin gene, α-globin gene, human growth hormone gene and the like. The target gene used in the present invention is not particularly limited, but a p21 gene (WAF1 / CIP1 gene) for stopping the cell cycle, a p53 gene for inducing cell death, and the like can also be handled by this method. .

The present invention also relates to a target gene transfer vector in which a target gene has been introduced into the above-described vector for gene transfer and / or the production of a gene-deficient non-human animal; And particularly to cells comprising an expression system capable of expressing a target gene. The introduction of all or a part of the DNA of the gene of interest into a cell is carried out by Davi.
(BASIC METHODS IN MOLECULAR BIOLOGY, 1986) and Sambrook et al. (MOLECULAR CLONING: A LABORATORY MANU
AL, 2nd Ed., Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, NY, 1989) and methods described in many standard laboratory manuals, such as calcium phosphate transfection, DEAE-dextran mediated transfection, transvection (t
ransvection), microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading (scrape loadi)
ng), ballistic introduction, infection and the like.

The above-mentioned cells are not particularly restricted but include bacterial prokaryotic cells such as Escherichia coli, Streptomyces, Bacillus subtilis, Streptococcus and Staphylococcus, fungal cells such as yeast and Aspergillus, Drosophila S2, Spodoptera. Insect cells such as Sf9, ES cells, fertilized eggs, L cells, CHO cells, COS cells, HeL
a cells, C127 cells, BALB / c3T3 cells (including mutants lacking dihydrofolate reductase, thymidine kinase, etc.), animal cells such as BHK21 cells, HEK293 cells, Bowes malignant melanoma cells, and plant cells. Can be.

The method for producing a transgenic non-human animal of the present invention includes the above-described vector for producing a transgenic and / or non-human gene-deficient animal and the above-mentioned cells, for example, a method for preparing a fertilized egg pronucleus. By microinjection of all or a part of the DNA for gene transfer and / or gene-defective non-human animal production vector, method of producing chimera after introducing the above DNA into ES cells, retrovirus having the above DNA Specific examples include a method of infecting and introducing a vector into an early embryo. As the non-human animal, a mouse,
Specific examples include non-human mammals such as rats, rabbits, pigs, goats, and cows, but are not limited thereto. The method for producing a transgenic non-human animal of the present invention is described below using the DNA
A method for producing a chimera after introduction of is described as an example.

For example, a series of units of the vector for producing a gene-introduced and / or gene-deficient non-human animal of the present invention,
For example, a promoter gene, a DNA encoding a fluorescent protein having a recombinase recognition sequence at both ends and a polyA signal fused downstream, a target gene, polyA
A targeting vector is prepared by incorporating genomic fragments (1 to 10b) before and after a site into which a target gene is to be introduced into unique sites located at both ends of DNA linked in the order of signals. At this time, the thymidine kinase (PGK-tk) gene of phosphoglycerokinase, the diphtheria toxin A fragment (DT-A) gene, the thymidine kinase of herpes simplex virus (HSV-t) are located at the 3 'downstream of the above series of units.
k) The presence of a negative selectable marker gene such as a gene is preferable because a recombinant in which the desired homologous recombination has not occurred in ES cells can be removed.

The above-mentioned targeting vector is linearized,
ES by electroporation method
The cells are introduced into the cells, homologous recombination is performed, and among the homologous recombinants, ES cells that have undergone homologous recombination with an antibiotic such as ganciclovia (GANC) are selected. It is also preferable to confirm whether the selected ES cells are the desired recombinant cells by Southern blotting or the like. The confirmed ES cell clone is microinjected into a mouse blastocyst, and the blastocyst is returned to a foster parent mouse to produce a chimeric mouse. By causing this chimeric mouse to intersect with a wild-type mouse, a transgenic mouse into which a foreign gene has been introduced in a site-specific manner can be produced. Further, as a method for confirming whether or not the transgenic mouse is the target, for example, RNA is isolated from the mouse obtained by the above method and examined by Northern blotting or the like, or the gene expression in the obtained mouse is determined. Examples thereof include a method of examining by a Western blot method, a fluorescence microscope and the like.

Next, a method for producing a gene-deficient non-human animal will be described by taking a knockout mouse as an example. Any method can be used as long as a knockout mouse that has lost the function of expressing the gene to be deleted can be produced. For example, using, as a probe, all or a part of the target gene, a mouse genomic DNA library is screened, the target gene of the genomic DNA is isolated, and the exon portion of the target gene and the introns surrounding the target gene are transfected. And / or a series of units of a vector for producing a gene-deficient non-human animal, for example, a promoter gene, a DNA encoding a fluorescent protein having a recombinase recognition sequence at both ends and a polyA signal fused downstream, a gene of interest, and a polyA signal In the order of To prepare a restrictor, ES target vectors produced by the electroporation method
Heterozygous mice obtained by crossing with wild-type mice by selecting ES cells that have been introduced into cells and undergoing homologous recombination, producing germ-line chimeric mice using this ES cell line, By crossing F1: the first generation of hybrids, a wild-type mouse having the same litter as a knockout mouse produced according to Mendel's law can be produced.

The cells obtained by using the target gene-introducing vector as described above, transgenic non-human animals, and cells, tissues or organs derived from such non-human animals can control the expression time of the target gene. In addition, since the target gene is introduced in a site-specific manner limited to one copy, it is useful for elucidating the mechanism of a genetic disease caused by expression of the target gene, developing therapeutics, and the like. As a method for expressing a target gene, for example, recombinase Cre when a loxP sequence is used as a recombinase recognition sequence, and recombinase FLP when a FRT sequence is used, are introduced into an expression vector. Can be specifically mentioned as a method for introducing a gene into cells or animal individuals. The method for introducing the recombinase gene expression vector into cells or animal individuals is not particularly limited. Examples of the method for introducing cells include electroporation, calcium phosphate coprecipitation, DEAE-dextran, lipofection, and gene transfection. Guns and the like can be mentioned, and as a method for introduction into animal individuals, Adeno-
Recombinase recombinant adenovirus such as cre, CM
Known methods such as a method of intravenously administering a recombinant cytomegalovirus or the like such as V-cre or the like can be specifically mentioned. In addition, by crossing the transgenic mouse of the present invention with a Cre-expressing transgenic mouse, a target gene can also be expressed. Furthermore, by injecting the recombinase gene expression vector or recombinase recombinant adenovirus in a site-specific manner, or by crossing the transgenic mouse of the present invention with a site-specific recombinase-expressing transgenic mouse, the target gene The expression can be induced site-specifically, or the expression of a posterior target gene at a localized site can be induced.

[0024]

The present invention will be described below with reference to examples.
The present invention is not limited by such embodiments. Example 1 [Construction of pCGFPtk Vector] The pCGFPtk vector was obtained by ligating the following mother nucleus, promoter gene, gene containing EGFP cDNA and SV40 polyA, and β-globin polyA. For preparation of the mother nucleus, refer to the literature (Cell 32, 1301-131).
1, 1983) using the plasmid pRH43 (see FIG. 2). The EcoRI site present in the plasmid pRH43 was digested and blunted, and a SalI linker (Stratagene) was inserted. In addition, Xho of plasmid pRH43
PGK-tk as a SalI / XhoI fragment at the I site
The gene (Cell 65, 1153-1163, 1991) was inserted in the direction shown in FIG. 2 (the direction opposite to the endogenous gene). Then removed by smoothing the HindIII / BamHI fragment containing the Kan ^R portion sandwiched by loxP sequences,
It has a loxP sequence at both ends and has a PGK-tk gene and AM
Those containing P ^R portion was used as a mother nucleus. As the promoter gene, the plasmid pCAGGS shown in FIG. 3 described in the literature (Gene 108, 193-20, 1991) was replaced with the restriction enzyme S.
1. Obtained by digesting with all and Xhol.
The 7 kb fragment CAG promoter (modified chichin)
β-actin promoter) was used.

A gene containing EGFP cDNA and SV40 polyA is pEGFP shown in FIG.
-N1 (Clontech) with restriction enzymes BamHI and Af
A 1 kb fragment obtained by digestion with II and blunting was used. As the β-globin poly A, a 0.5 kb fragment obtained by digesting the above plasmid pCAGGS with the restriction enzymes PstI / XhoI and blunting it was used.
Rabbit β-globin poly A consisting of the 3 ′ flanking sequence of globin was previously cut at the BglII site and blunt-ended, and the plasmid pUK21 (Ge
ne 03971, 189-194, 1991).
Globin is 5 'SalI → β-globin → PstI3'
Β-globin polyA obtained by cleaving at a SalI / XhoI site using a clone in the orientation of 1. and a multicloning site upstream and downstream of the β-globin polyA.
A 5 kb fragment was used. Then, as shown in FIG. 6, 1. was placed at the SalI site of the mother nucleus having loxP sequences at both ends.
A 7 kb SalI / XhoI fragment (CAG promoter) was inserted, a 0.5 kb fragment containing a multicloning site and β-globin polyA was inserted into the XhoI site, and EGFP cDNA was inserted into both ends of the loxP sequence.
And a gene containing polyA were ligated in the forward direction to obtain 8.5 kb.
PCGFPtk vector was constructed (see FIG. 1).

Example 2 [Introduction of Target Gene and Expression of GFP] A lethal gene p73L derived from a mouse to which an HA tag was added as a target gene was inserted into the multicloning site of the prepared pCGFPtk vector (Biochemical
and Biophysical Research Communications 248, 603-6
07, 1998) containing the MluI / PstI fragment. PCGFPtk into which the target gene has been introduced
2 μg of the vector (hereinafter referred to as “pCGFP-tk-geneA”) was introduced into COS7 cells (1 × 10 ⁵ cells) in a 24-well dish by a lipofection method. After the introduction, the cells were cultured in a DMEM (Dulbecco's modified MEM) medium containing 15% FCS (fetal bovine serum) at 37 ° C. for 24 hours, and luminescence of GFP was confirmed by a fluorescence microscope. As a result, luminescence of GFP was confirmed in about 70% of the cells.

Example 3 (Confirmation of operation of Cre-loxP) pCGFP-tk-geneA prepared in Example 2
2 μg was introduced into COS7 cells (1 × 10 ⁵ ) by the lipofection method in the same manner as described above in the presence or absence of CMV-Cre or Adeno-Cre. After introduction, DM containing 15% FCS (fetal calf serum)
After culturing at 37 ° C. for 48 hours in an EM (Dulbecco-modified MEM) medium, the cells were collected and lysed, and subjected to Western blotting according to a standard method. An anti-HA antibody was used for detection. FIG. 7 shows the results. In FIG. 7, lane 1 is pCGF
Lane 2 is CMV-Cre only for P-tk-geneA.
Lane 3, Lane 3 only with Adeno-Cre, Lane 4 with pCGFP-tk-geneA and CMV-Cre, Lane 5 with pCGFP-tk-geneA and Adeno
-Shows the results of the introduction of Cre, and as shown in lanes 4 and 5, only when Cre expression was involved, p
It was confirmed that the expression of the target gene was induced from CGFP-tk-geneA.

Example 4 (Preparation of transgenic mouse and expression analysis in vitro) pCGFP-tk-geneA prepared in Example 2
With 10 mM Tris (pH 7.5) and 0.25 m
M was suspended in a buffer containing EDTA at 4 to 8 ng / μl. Using an inverted differential interference microscope (“IX70” manufactured by Olympus Corporation), microinjection was performed into fertilized mouse mice under a visual field of 200 times, and transplanted into the oviduct of a pseudopregnant (foster mother) female mouse to produce a chimeric mouse. By crossing the chimeric mouse with a wild-type female mouse (C57BL / 6), a transgenic mouse (F1) into which the target gene was introduced was obtained.

Example 5 (Detection of Target Gene by Southern Blot Method) Mother mouse (lane 1), individual symbol J (lane 2),
10 μg of genomic DNA obtained by digesting DNA prepared from the tail of the F1 mouse of K (lane 3) and L (lane 4) with EcoRI was electrophoresed on a 1% agarose gel, and was subjected to a turbo blotter system (Schleicher
and Schunell). Next, a genomic fragment fixed on the membrane and a Prime-It II random primer labeling kit (Stratagene)
Full-length cD of ³² P-labeled target gene A synthesized by
Hybridization with the NA probe was performed, and the foreign transgene was detected by autoradiography.
The results of the Southern blot are shown in FIG. Lane 2 of FIG.
As shown in FIG. 3, the target gene A is about 1 in the J and K strains.
It was confirmed that 5-20 copies had been introduced, and it was revealed that the mouse was a transgenic mouse

The target gene A has an ability to induce apoptosis and is lethal by ordinary transgenic techniques. This means that the transgenic mouse can be used even if the target gene A directly linked to the CAG promoter, CMV promoter or PGK promoter is used. Was not obtained at all, and when the transgenic mouse into which the target gene was introduced and the systemically expressed Cre transgenic mouse were crossed, no F1 individual having both transgenes was obtained (84 animals). (Medium, 0). In the present invention, the p gene in which the target gene A is linked downstream of the GFP gene sandwiched by the loxP sequence
When CGFP-tk-geneA was used, as shown below, pCGFP-tk-geneA expressed only GFP in animal cells and did not express downstream target gene A, but it was accompanied by recombinase Cre expression. And G
It can be seen that the FP gene is removed and the downstream target gene A is expressed for the first time.

Example 6 (Expression of GFP in each organ of transgenic mouse) Transgenic mice of the above two strains J and K were dissected by a conventional method, and UV lamps (BLAK-RAY LAMP, I
ONG WAVE UV-366nm, Model UVL-56, San Gabriel, CA)
Under irradiation, luminescence of GFP in each organ of the transgenic mouse was visually observed. In addition, a wild-type mouse (C57BL / 6) was used as a control. As a result, intense luminescence was observed in the heart, pancreas, skeletal muscle, and peritoneum of these transgenic mice. In addition, when cells derived from organs of each transgenic mouse were isolated and subjected to flow cytometry, GFP was found in a part (5 to 10%) of thymus and spleen cells among organs that could not be observed with the naked eye. Light emission was observed. However, no GFP emission was observed in control mice in all of these organs or cells. According to this confirmation method, it is possible to easily select a strain having GFP expression in a target organ from a plurality of obtained individuals. FIG. 9 shows that the transgenic mouse of J strain (male) was
Fetal age 1 obtained by crossing with 57BL / 6 mouse (female)
The results on 4.5 day fetal fibroblasts are shown. In FIG. 9, Data001 is a littermate control, and Data002 is a fetal fibroblast derived from a transgenic mouse. F
As shown in the CS / SSC, there was no difference in the appearance of each cell group, while only about 95% of the transgene-bearing fetal fibroblasts were positive for GFP luminescence. Fetal fibroblasts are frequently used materials to reproduce phenomena at an individual level in vitro, and fetal fibroblasts having the transgene of the present invention can induce the expression of a target gene in a time-specific manner. Useful in Further, the present invention can be similarly applied to various cells established from desired organs other than fetal fibroblasts.

Example 7 (Expression of Target Gene in Fetal Fibroblasts Derived from Transgenic Mice) Next, the fetal fibroblasts were infected with Adeno-Cre, and 24 hours later, immunoprecipitation was performed with a mouse anti-HA antibody. Was. Detection was performed using a rabbit anti-HA antibody. Figure 1 shows the results
0 is shown. In FIG. 10, lane 1 shows control fetal fibroblasts infected with Adeno-Cre, lane 2 shows transgenic mouse-derived fetal fibroblasts not infected with Adeno-Cre, and lane 3 shows transgenic mice. Aden against mouse-derived fetal fibroblasts
From the results of immunoprecipitation of those infected with o-Cre, GF
Adeno-Cr against fetal fibroblasts showing P emission
e, it was confirmed that the target gene was expressed by infection. At this time, GFP luminescence had completely disappeared.

[0033]

According to the present invention, gene transfer can be carried out by limiting the target gene such as a lethal gene to one copy, selecting a site to be introduced, and controlling the expression time of the target gene. And / or a vector for producing a gene-deficient non-human animal can be constructed, and cells or transgenic non-human animals produced by using such a gene-introducing and / or gene-deficient non-human animal producing vector can be introduced. Since the expression site of the gene can be easily detected and the target gene can be expressed at a desired time, it can be applied to the elucidation of the mechanism of genetic diseases and the development of therapeutics due to the expression of the target gene. Can be expected.

[Brief description of the drawings]

FIG. 1 is a schematic diagram of the vector of the present invention for producing a gene and / or a gene-deficient non-human animal, and the vector when a recombinase Cre is allowed to act thereon.

FIG. 2 is an explanatory diagram showing production of a mother nucleus constituting the vector for gene introduction and / or production of a gene-deficient non-human animal of the present invention.

FIG. 3 is an explanatory diagram showing the production of a promoter portion constituting a vector for gene introduction and / or production of a gene-deficient non-human animal of the present invention.

FIG. 4 shows EGFP cDNA and S constituting the vector for gene transfer and / or production of a gene-deficient non-human animal of the present invention.
FIG. 4 is an explanatory diagram showing the production of a gene containing V40 polyA.

FIG. 5 is an explanatory diagram showing the production of β-globin poly A containing a multicloning site constituting the vector for gene introduction and / or production of a gene-deficient non-human animal of the present invention.

FIG. 6 is an explanatory diagram showing the construction of a vector for producing a gene and / or a gene-deficient non-human animal of the present invention.

FIG. 7 is a view showing the result of examining the expression of a target gene in cells into which the transgene of the present invention has been introduced by Western blotting.

FIG. 8 is a view showing the result of examining the presence of a target gene in the transgenic mouse of the present invention by Southern blotting.

FIG. 9 shows the results of FACS analysis of GFP luminescence in fetal fibroblasts derived from the transgenic mouse of the present invention.

FIG. 10 is a diagram showing the results of examining the expression of a target gene in fetal fibroblasts derived from the transgenic mouse of the present invention by immunoprecipitation.

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Claims (16)

[Claims] Claims: 1. A vector for introducing a gene of interest and / or for producing a gene-deficient non-human animal, the function of which can control the expression time of the gene of interest, and the function of the gene of interest. A vector having a function of early detection of an expression site. 2. The vector according to claim 1, wherein the vector has a function of limiting the target gene to one copy and introducing it site-specifically. 3. A vector for gene transfer and / or production of a gene-deficient non-human animal, comprising a promoter gene, a DNA encoding a fluorescent protein fused with a polyA signal having a recombinase recognition sequence at both ends, and a target gene on the upstream side. 3. The vector according to claim 1, which is a vector containing DNA linked in the order of poly A signal having a multi-cloning site for introduction. 4. The vector according to claim 3, wherein the recombinase recognition sequence is a loxP or FRT sequence. 5. The poly A signal derived from the β-galactosidase gene, SV40, β-globin gene, α-globin gene or human growth hormone gene, wherein the poly A signal is derived from the poly A signal. Vector. 6. The fluorescent protein is GFP or EGFP.
(Enhanced GFP), EYFP (Enhanced Yellow Fluore)
scent Protein), ECFP (enhanced CYAN fluorescen)
t protein) or DsRed. The vector according to any one of claims 3 to 5, wherein the vector is DsRed. 7. The vector according to claim 1, comprising a negative selectable marker gene. 8. The vector according to claim 7, wherein the negative selectable marker gene is thymidine kinase. 9. A vector for introducing a gene of interest, wherein the gene of interest has been introduced into the vector for gene introduction and / or the production of a gene-deficient non-human animal according to any one of claims 1 to 8. 10. The target gene transfer vector according to claim 9, wherein the target gene is a lethal gene. 11. A cell into which all or a part of the DNA of the gene transfer vector according to claim 9 or 10 has been introduced. 12. A DNA of the whole or a part of the target gene transfer vector according to claim 9 or 10, and claim 1.
A method for producing a transgenic or genetically deficient non-human animal, comprising using the cell of claim 1. 13. A non-human transgenic or gene-deficient animal obtained by the method of claim 12. 14. The transgenic or genetically deficient non-human animal according to claim 13, wherein the non-human animal is a mouse, rat, rabbit, pig, goat, or cow. 15. A cell, tissue or organ obtained from the transgenic or genetically deficient non-human animal according to claim 13 or 14. 16. A target gene obtained by treating the cell according to claim 11, the transgenic or deficient non-human animal according to claim 13 or 14, or the cell, tissue or organ according to claim 15 with a recombinase. A cell, tissue, organ or transgenic or gene-deficient non-human animal in which is expressed.