JP6327712B2 - Method for producing transgenic non-human animal and method for producing genetically modified non-human animal - Google Patents

Description

  The present invention relates to a transgenic non-human animal and a production method thereof, a genetically modified non-human animal and a production method thereof, and an expression vector production vector and an expression vector.

  Currently, knock-out mice prepared using ES cells are widely used for in vivo gene function analysis. In addition, a conditional knockout mouse using a recombinase / recombinase recognition sequence system such as a Cre / loxP system is used for the analysis of a time-specific or tissue-specific gene function (for example, see Non-Patent Document 1). .)

Nagy A., Cre recombinase: the universal reagent for genome tailoring, Genesis, 26 (2), 99-109, 2000.

  However, the production of knockout mice and conditional knockout mice is very expensive, and requires special technologies such as ES cell culture technology and chimera mouse production technology, as well as dedicated facilities. It is not a general analytical tool that can be carried out even in research facilities.

  In addition, the creation of knockout mice and conditional knockout mice takes about 2 to 3 years even if they proceed smoothly, and individual problems arise in each study. Twice as much time is required. For this reason, establishment of a novel method capable of analyzing a time-specific or tissue-specific gene function in a short time, with high efficiency and at low cost is desired.

  In addition, ES cells are required for the production of a conditional knockout mouse. However, since most ES cells that can currently differentiate into germline are ES cells derived from 129 mice, other than 129 mice There is a problem that it is impossible to analyze the function of a gene whose specific phenotype is confirmed only in a specific strain of mice.

  Therefore, the present invention provides a method for producing a non-human animal, which can more easily analyze a time-specific or tissue-specific gene function and can be applied to an animal in which ES cells are not established. The purpose is to provide. Another object of the present invention is to provide a non-human animal produced by the above production method, an expression vector used in the production method, and a vector for producing the expression vector.

The present invention is as follows.
(1) An expression vector having a first recombinase recognition sequence, a wild-type target gene, and a second recombinase recognition sequence in this order, and a phenotype different from the wild type having a mutation in the target gene A non-human animal that is a fertilized egg of a non-human animal in which no ES cell has been established, or a non-human animal that lacks the target gene and exhibits a phenotype different from the wild type , the ES cell A transgenic non-human animal whose phenotype is rescued, comprising the steps of: introducing into a fertilized egg of a non-human animal in which the phenotype is not established; and culturing the fertilized egg to obtain a transgenic non-human animal. Production method.
(2) a transgenic non-human animal having a transgene having a first recombinase recognition sequence, a wild-type target gene, and a second recombinase recognition sequence in this order, wherein the phenotype is wild type; The transgenic non-human animal, wherein the endogenous gene of interest has a mutation or is deleted, and the non-human animal is an animal in which ES cells are not established.
(3) Recombinase is caused to act in a time-specific or tissue-specific manner on the transgenic non-human animal described in (2), and the introduced wild-type target gene is deleted in a time-specific or tissue-specific manner A method for producing a genetically modified non-human animal comprising a step, wherein the target gene is mutated or deleted in a time-specific or tissue-specific manner.
(4) A transgenic non-human animal having a transgene having a first recombinase recognition sequence, a wild-type target gene, and a second recombinase recognition sequence in this order, wherein the endogenous target gene is: A gene having a mutation or deletion, wherein the non-human animal is an animal in which ES cells are not established, and the target gene in the transgene is deleted in a time-specific or tissue-specific manner Modified non-human animal.
(5) A vector for producing an expression vector used in the production method according to (1), wherein a first recombinase recognition sequence, a promoter, a rescue cassette, and a second recombinase recognition sequence are arranged in this order. A vector wherein the rescue cassette has a multiple cloning site.
(6) The vector according to (5), wherein the rescue cassette has a multicloning site and a fluorescent protein gene in this order.
(7) The vector according to (5), wherein the rescue cassette has a multicloning site, a base sequence that enables polycistronic expression, and a fluorescent protein gene in this order.
(8) The vector according to (5), wherein the rescue cassette has a fluorescent protein gene, a base sequence that enables polycistronic expression, and a multicloning site in this order.
(9) An expression vector used in the production method according to (1) , comprising a first recombinase recognition sequence, a promoter, a rescue cassette, and a second recombinase recognition sequence in this order, An expression vector in which the rescue cassette has a wild-type target gene.
(10) The expression vector according to (9), wherein the rescue cassette has a gene encoding a fusion protein of a protein encoded by the wild-type target gene and a fluorescent protein.
(11) The expression vector according to (9), wherein the rescue cassette has the wild-type target gene, a base sequence that enables polycistronic expression, and a fluorescent protein gene in this order.
(12) The expression vector according to (9), wherein the rescue cassette has a fluorescent protein gene, a base sequence that enables polycistronic expression, and the wild-type target gene in this order.
(13) A vector for producing an expression vector used in the production method according to (1), wherein a promoter, a first recombinase recognition sequence, a rescue cassette, a second recombinase recognition sequence, and a first A fluorescent protein gene in this order, and the rescue cassette has a multicloning site.
(14) The vector according to (13), wherein the rescue cassette has a multicloning site and a second fluorescent protein gene in this order.
(15) The vector according to (13), wherein the rescue cassette has a multicloning site, a base sequence that enables polycistronic expression, and a second fluorescent protein gene in this order.
(16) The vector according to (13), wherein the rescue cassette has a second fluorescent protein gene, a base sequence that enables polycistronic expression, and a multicloning site in this order.
(17) An expression vector used in the production method according to (1) , comprising a promoter, a first recombinase recognition sequence, a rescue cassette, a second recombinase recognition sequence, and a first fluorescent protein gene In this order, and the rescue cassette has a wild-type target gene.
(18) The expression vector according to (17), wherein the rescue cassette has a gene encoding a fusion protein of a protein encoded by the wild-type target gene and a second fluorescent protein.
(19) The expression vector according to (17), wherein the rescue cassette has the wild-type target gene, a base sequence that enables polycistronic expression, and a second fluorescent protein gene in this order. .
(20) The expression vector according to (17), wherein the rescue cassette has a second fluorescent protein gene, a base sequence enabling polycistronic expression, and the wild-type target gene in this order. .

  According to the present invention, there is provided a method for producing a non-human animal that can more easily analyze a time-specific or tissue-specific gene function and can be applied to an animal in which ES cells are not established. be able to. Moreover, the non-human animal manufactured by said manufacturing method, the expression vector used for said manufacturing method, and the vector for the said expression vector manufacture can be provided.

(A)-(d) is a schematic diagram which shows the structure of the vector (1-1)-(1-4) for expression vector manufacture, respectively. In the figure, “MCS” means a multiple cloning site, and “Ires” means an Ires sequence. (A)-(d) is a schematic diagram which shows the structure of expression vector (1-1)-(1-4). In the figure, “fusion fluorescent protein gene” means a gene encoding a fusion protein of a protein encoded by a wild-type target gene and a fluorescent protein. (A)-(d) is a schematic diagram which shows the structure of the vector (2-1)-(2-4) for expression vector manufacture. (A)-(d) is a schematic diagram which shows the structure of expression vector (2-1)-(2-4). (A) And (b) is the fluorescence micrograph which image | photographed the ES cell of the control group and the test group, respectively.

[Method for producing transgenic non-human animal]
In one embodiment, the present invention provides an expression vector having a first recombinase recognition sequence, a wild-type target gene, and a second recombinase recognition sequence in this order, and the target gene has a mutation and a wild-type A non-human animal exhibiting a phenotype different from that of the target or a non-human animal lacking the target gene and exhibiting a phenotype different from the wild type, and introducing the fertilized egg into a transgenic egg Obtaining a non-human animal, and a method for producing a transgenic non-human animal whose phenotype is rescued.

  By the production method of the present embodiment, a non-human animal having a mutation in the target gene and showing a phenotype different from the wild type, or a non-human animal lacking the target gene and showing a phenotype different from the wild type Then, a wild-type target gene is introduced, and the above phenotype is rescued, so that a transgenic non-human animal exhibiting the wild-type phenotype can be produced.

  As described later, the time-specific or tissue-specific function of the target gene is analyzed by deleting the time-specific or tissue-specific wild-type target gene introduced into the transgenic non-human animal. can do.

  Transgenic non-human animals do not require site-specific homologous recombination, etc., and are much easier than knockout non-human animals and conditional knock-out non-human animals in that they can be generated without using ES cells. Can be produced. Since the production method of the present embodiment can be carried out if a transgenic non-human animal can be produced, it can be carried out more easily and can also be applied to animals in which ES cells have not been established.

(Non-human animals)
The non-human animal that is a target for producing a transgenic non-human animal by the production method of the present embodiment is not particularly limited, and examples thereof include mice, rats, rabbits, pigs, cows, monkeys, chickens, and the like. However, it is necessary to be a non-human animal that has been clarified to exhibit a phenotype different from the wild type by having a mutation in the target gene or by deleting the target gene. In the production method of this embodiment, by introducing a wild-type target gene into such a non-human animal, a phenotype is rescued, and a transgenic non-human animal exhibiting the wild-type phenotype is produced.

  The production method of the present embodiment can be applied to, for example, many disease model mice obtained by conventional research. This makes it possible to analyze time-specific or tissue-specific gene functions in a short time with respect to the causative genes of many disease model mice that have not yet been elucidated.

(Recombinase recognition sequence)
In the present specification, the term “recombinase recognition sequence” refers to a recombinase recognition sequence in a so-called recombinase / recombinase recognition sequence system that utilizes the phenomenon that a specific recombinase recognizes a recombinase recognition sequence and causes DNA recombination at that portion. means. In the present specification, the term “base sequence” may mean a nucleic acid comprising the base sequence.

  Examples of the recombinase / recombinase recognition sequence system include a Cre recombinase derived from bacteriophage P1, a Cre / loxP system using a 34 base pair loxP sequence recognized by Cre recombinase, a FLP recombinase derived from yeast, and a FLP recombinase. FLP / FRT system using FRT sequence to be used, R recombinase derived from Zygosaccharomyces rouxii, R / RS system using RS sequence recognized by R recombinase, and the like. Therefore, examples of the recombinase recognition sequence used in the production method of this embodiment include a loxP sequence, an FRT sequence, and an RS sequence.

  As the loxP sequence, in addition to the base sequence represented by 5′-ATAACTTCGTATATAGCATACATTATACGAAGTTAT-3 ′ (SEQ ID NO: 1), for example, Siegel RW, et al., Using an in vivo phagemid system to identify non-compatible loxP sequences, FEBS Lett., 505, 467-473, 2001. 5′-ATAACTTCGTATAGTATACCATTATACGAAGTTAT-3 ′ (lox511, SEQ ID NO: 2), 5′-ATAACTTCGTATAGATATACTTTATACGAAGTTAT-3ATGATACTATAGTAC Mutant loxP sequences such as −3 ′ (loxFAS, SEQ ID NO: 4) can be used.

  As the FRT sequence, for example, a base sequence such as 5'-GAAGTTCCCTATTCTCTGAAAAGTATAGGAACTTC-3 '(SEQ ID NO: 5) can be used.

  As the RS sequence, for example, a base sequence such as 5'-TTGATGAAGAAAATAACGTATTCTTTCATCAA-3 '(SEQ ID NO: 6) can be used.

  Even if a recombinase recognition sequence is present on the genome, recombination does not occur unless a specific recombinase is present. Therefore, for example, by mating a non-human animal that expresses recombinase in a time-specific or tissue-specific manner with a non-human animal of the same species into which the recombinase recognition sequence has been introduced, recombination with the recombinase is performed in the F1 animal. It can be time-specific or tissue-specific.

(Expression vector)
The expression vector used in the production method of the present embodiment has a first recombinase recognition sequence, a wild-type target gene, and a second recombinase recognition sequence in this order, and allows the wild-type target gene to be expressed. There is no particular limitation as long as it is possible. By introducing this expression vector into the above-mentioned non-human animal showing a phenotype different from that of the wild type and expressing the target gene of the wild type, the phenotype of the non-human animal is rescued, and the wild type phenotype is changed. The transgenic non-human animals shown can be obtained.

  The first recombinase recognition sequence and the second recombinase recognition sequence are preferably arranged so that the wild-type target gene is excised when the above-described recombinase / recombinase recognition sequence system is activated. Therefore, it is preferable that the first recombinase recognition sequence and the second recombinase recognition sequence are arranged in the same direction across the wild-type target gene. Thereby, the introduced wild-type target gene can be deleted in the tissue in which the recombinase is allowed to act.

  The expression vector is not particularly limited. For example, plasmids derived from E. coli such as pBR322, pBR325, pUC12, and pUC13; plasmids derived from Bacillus subtilis such as pUB110, pTP5, and pC194; plasmids derived from yeast such as pSH19 and pSH15; Bacteriophages; viruses such as adenoviruses, adeno-associated viruses, lentiviruses, vaccinia viruses, baculoviruses; and modified vectors thereof.

  In the above expression vector, the promoter for expression of the target gene is not particularly limited, and for example, EF1α promoter, SRα promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, HSV-tk promoter, etc. may be used. Can do.

  The expression vector may further have an enhancer, a splicing signal, a poly A addition signal, a selection marker, an origin of replication, and the like.

  The expression vector is injected into the pronucleus pronucleus collected from a non-human animal using a microcapillary or the like under a microscope, and the fertilized egg is transplanted into the oviduct of the recipient animal. Subsequently, the target transgenic non-human animal can be obtained by selecting individuals from which the gene of interest has been incorporated into somatic cells and germ cells from the individuals that have occurred.

  Moreover, a heterozygote can be obtained from an F1 animal obtained by mating an individual in which a target gene is incorporated into a germ cell with a wild-type non-human animal. Furthermore, the above-mentioned heterozygote can be crossed and a homozygote can be obtained from the obtained F1 animal.

  The selection of heterozygote or homozygote can be performed, for example, by screening chromosomal DNA collected from the F1 animal by Southern hybridization or PCR.

[Transgenic non-human animals]
In one embodiment, the present invention provides a transgenic non-human animal produced by the production method described above. The transgenic non-human animal of the present embodiment can delete a wild-type target gene introduced in the tissue by allowing recombinase to act on the target tissue. By deleting the wild-type target gene in a time-specific or tissue-specific manner, the time-specific or tissue-specific function of the target gene can be analyzed.

[Method for producing genetically modified non-human animal]
In one embodiment, the present invention causes the above-described transgenic non-human animal to react with recombinase in a time-specific or tissue-specific manner and lacks the introduced wild-type target gene in a time-specific or tissue-specific manner. And a method for producing a genetically modified non-human animal in which the target gene is mutated or deleted in a time-specific or tissue-specific manner.

  As described above, the above-described transgenic non-human animal originally has a mutation in the target gene or lacks the target gene, and the non-human animal that showed a phenotype different from the wild type is The phenotype was rescued by the introduction of the wild-type target gene, indicating a wild-type phenotype.

  Effect of mutation or deletion of the target gene originally possessed by the non-human animal by the time-specific or tissue-specific deletion of the introduced wild-type target gene by the production method of the present embodiment However, it is possible to produce a non-human animal that has been time-specific or tissue-specific.

  Therefore, the production method of this embodiment can produce a non-human animal in which the target gene is mutated or deleted in a time-specific or tissue-specific manner. As described above, such a non-human animal can be easily produced even in an animal in which ES cells have not been established. This non-human animal can be used to analyze the time-specific or tissue-specific function of the target gene.

  As the recombinase, the recombinase in the recombinase / recombinase recognition sequence system described above can be used, and the recombinase corresponding to the recombinase recognition sequence introduced into the transgenic non-human animal is used.

  Examples of the recombinase include Cre recombinase derived from bacteriophage P1, FLP recombinase derived from yeast, R recombinase derived from Digosaccharomyces roxy and the like.

  As the Cre recombinase, Cre-ER protein that is a fusion protein of Cre recombinase and mutant estrogen receptor may be used. The Cre-ER protein is usually present in the cytoplasm, but translocates into the nucleus by binding to the estrogen derivative tamoxifen and causes recombination to the loxP sequence. By utilizing this, it is possible to control the time or tissue in which Cre recombinase is allowed to act by systemically or locally administering tamoxifen to the transgenic non-human animal.

  Examples of a method for allowing recombinase to act in a time-specific or tissue-specific manner include, for example, mating the above-described transgenic non-human animal with a transgenic non-human animal that expresses the recombinase under the control of a time-specific or tissue-specific promoter. The method of doing is mentioned. In this case, in the obtained F1 animal, the target gene is deleted in a time-specific or tissue-specific manner only at a site where a time-specific or tissue-specific promoter works. In addition, by appropriately selecting a promoter for expressing recombinase, a desired time-specific or tissue-specific deletion of the target gene can be induced.

  Specific examples of promoters that enable time-specific expression include, for example, the Neuron specific enolase gene (expressed in neurons after 3 days of birth), the Pcp2 gene (expressed in cerebellar Purkinje cells after 6 days of birth) And the like. Specific examples of promoters that allow tissue-specific expression include, for example, albumin gene (specifically expressed in hepatocytes), insulin gene (specifically expressed in pancreatic β cells), Nestin gene (neural stem cells) And a promoter expressed specifically in glial cells).

  As another method for causing the recombinase to act in a time-specific or tissue-specific manner, for example, a recombinase expression vector or a recombinase protein can be directly administered to the above-described transgenic non-human animal. Administration may be systemic administration or local administration. By adjusting the time when the recombinase expression vector or the recombinase protein is administered or the tissue to which the recombinase protein is administered, the time or tissue where the recombinase acts can be controlled.

[Gene modified non-human animals]
In one embodiment, the present invention provides a genetically modified non-human animal produced by the production method described above.

  As described above, in the non-human animal of this embodiment, the influence of the mutation or deletion of the target gene originally possessed by the non-human animal is manifested in a time-specific or tissue-specific manner. Therefore, it is possible to analyze the time-specific or tissue-specific function of the target gene using the non-human animal of this embodiment.

[Vector for production of expression vector (1)]
In one embodiment, the present invention is a vector for producing an expression vector used in the above-described method for producing a transgenic non-human animal, comprising a first recombinase recognition sequence, a promoter, a rescue cassette, And a recombinase recognition sequence in this order, and the rescue cassette has a multicloning site (hereinafter sometimes referred to as “vector for expression vector production (1-1)”).

  Fig.1 (a) is a schematic diagram which shows the structure of the vector (1-1) for expression vector manufacture. In the figure, “first recognition sequence” means a first recombinase recognition sequence, “second recognition sequence” means a second recombinase recognition sequence, “MCS” means a multiple cloning site, The same applies hereinafter. The vector for producing an expression vector (1-1) has a mutation in the target gene or a deletion of the target gene by incorporating the wild type target gene into the multicloning site of the rescue cassette. Is suitable for producing a vector expressing a wild-type target gene for rescue of a phenotype of a non-human animal exhibiting a different phenotype.

  The first recombinase recognition sequence, the promoter, and the second recombinase recognition sequence are the same as described above.

  Since the vector for expression vector production (1-1) has a multicloning site in the rescue cassette, the above vector can be produced by easily introducing a wild-type target gene.

  The expression vector production vector (1-1) may further have an enhancer, a splicing signal, a poly A addition signal, a selection marker, an origin of replication, and the like.

  The rescue cassette of the expression vector production vector (1-1) may have a multicloning site and a fluorescent protein gene in this order (hereinafter referred to as “expression vector production vector (1-2)”). May be.)

  FIG.1 (b) is a schematic diagram which shows the structure of the vector (1-2) for expression vector manufacture. The expression vector production vector (1-2) is constructed such that when a wild-type target gene is introduced into a multicloning site, an expression vector of a fusion protein of the wild-type target gene product and a fluorescent protein is obtained. It is preferable. The transgenic non-human animal into which the expression vector is introduced emits fluorescence derived from the fluorescent protein at the site where the wild-type target gene is expressed.

  Therefore, for example, by detecting the fluorescence using a fluorescence microscope, it can be easily determined whether or not the wild-type target gene is expressed.

  The fluorescent protein gene is not particularly limited. For example, green fluorescent protein (GFP), red fluorescent protein (DsRed, Clontech), yellow fluorescent protein (YFP), blue fluorescent protein (BFP), cyan fluorescent protein (CFP) , TagRFP (Evogen), TagGFP2 (Evogen), and the like.

  The rescue cassette of the expression vector production vector (1-1) may have a multicloning site, a base sequence that enables polycistronic expression, and a fluorescent protein gene in this order (hereinafter, It may be referred to as “vector for producing expression vector (1-3)”.

  FIG.1 (c) is a schematic diagram which shows the structure of the vector (1-3) for expression vector manufacture. In the figure, a case where an Ires sequence is used as a base sequence that enables polycistronic expression is shown, and “Ires” in the figure means an Ires sequence, and the same applies hereinafter. The fluorescent protein gene is the same as the expression vector production vector (1-2).

(Base sequence enabling polycistronic expression)
Polycistronic expression means that a plurality of proteins are expressed from one mRNA. Examples of the base sequence that enables polycistronic expression include an Ires sequence, a base sequence encoding a 2A peptide, and the like.

  The Ires sequence means a base sequence encoding an internal ribosome entry site. If an Ires sequence is present in the middle of mRNA, a ribosome binds to the sequence and translation can be started therefrom. The Ires sequence is not particularly limited, and examples thereof include an Ires sequence of encephalomyocarditis virus (ECMV).

  A base sequence encoding 2A peptide is also known as a base sequence that enables polycistronic expression. When a base sequence encoding 2A peptide is present in mRNA, elongation of the polypeptide chain is inhibited and cleaved by a mechanism called ribosome skipping at the time of translation. As a result, two types of proteins, the protein encoded on the upstream side of the base sequence encoding the 2A peptide and the protein encoded on the downstream side, are translated. Examples of the base sequence encoding 2A peptide include a base sequence encoding 2A peptide of foot-and-mouth disease virus.

  The expression vector production vector (1-3) is constructed so that when a wild-type target gene is introduced into a multicloning site, an expression vector that expresses both the wild-type target gene and the fluorescent protein gene is obtained. Preferably it is.

  Such an expression vector is effectively used, for example, when the wild-type target gene product cannot function in the form of a fusion protein with a fluorescent protein. The transgenic non-human animal into which the expression vector is introduced emits fluorescence derived from the fluorescent protein at the site where the wild-type target gene is expressed.

  Therefore, for example, by detecting the fluorescence using a fluorescence microscope, it can be easily determined whether or not the wild-type target gene is expressed.

  The rescue cassette of the expression vector production vector (1-1) may have a fluorescent protein gene, a base sequence that enables polycistronic expression, and a multicloning site in this order (hereinafter, It may be referred to as “vector for producing expression vector (1-4)”.

  FIG.1 (d) is a schematic diagram which shows the structure of the vector (1-4) for expression vector manufacture. In the figure, a case where an Ires sequence is used as a base sequence that enables polycistronic expression is shown. The fluorescent protein gene is the same as described above. The vector for producing an expression vector of the present embodiment is obtained by replacing a fluorescent protein gene and a multicloning site in the vector for producing an expression vector (1-3), and functionally, the vector for producing an expression vector (1- Same as 3).

[Expression vector (1)]
In one embodiment, the present invention has a first recombinase recognition sequence, a promoter, a rescue cassette, and a second recombinase recognition sequence in this order, and the rescue cassette has a wild-type target gene. An expression vector (hereinafter sometimes referred to as “expression vector (1-1)”) is provided.

  Fig.2 (a) is a schematic diagram which shows the structure of an expression vector (1-1). The expression vector (1-1) can be suitably used for the production of the above-described transgenic non-human animal.

  The expression vector (1-1) can be obtained by introducing a wild-type target gene into the multiple cloning site of the rescue cassette of the above-described expression vector production vector (1-1).

  The first recombinase recognition sequence, the promoter, and the second recombinase recognition sequence are the same as those in the above-described expression vector production vector (1-1).

  The rescue cassette of the expression vector (1-1) may have a gene encoding a fusion protein of a protein encoded by the wild-type target gene and a fluorescent protein (hereinafter referred to as “expression vector (1- 2) ".

  FIG. 2B is a schematic diagram showing the structure of the expression vector (1-2). In the figure, “fusion fluorescent protein gene” means a gene that encodes a fusion protein of a protein encoded by a wild-type target gene and a fluorescent protein, and so on. The expression vector (1-2) is obtained by introducing a wild-type target gene into the multicloning site of the rescue cassette of the above-described expression vector production vector (1-2).

  The fluorescent protein gene is the same as the above-described expression vector production vector (1-2).

  The transgenic non-human animal into which the expression vector (1-2) has been introduced emits fluorescence derived from the fluorescent protein at the site where the wild-type target gene is expressed.

  Therefore, for example, by detecting the fluorescence using a fluorescence microscope, it can be easily determined whether or not the wild-type target gene is expressed.

  The rescue cassette of the expression vector (1-1) may have the wild-type target gene, a base sequence that enables polycistronic expression, and a fluorescent protein gene in this order (hereinafter, It may be referred to as “expression vector (1-3)”.

  FIG.2 (c) is a schematic diagram which shows the structure of an expression vector (1-3). In the figure, a case where an Ires sequence is used as a base sequence that enables polycistronic expression is shown. The expression vector (1-3) can be obtained by introducing a wild-type target gene into the multiple cloning site of the rescue cassette of the above-described expression vector production vector (1-3).

  The transgenic non-human animal into which the expression vector (1-3) has been introduced emits fluorescence derived from the fluorescent protein at the site where the wild-type target gene is expressed.

  Therefore, for example, by detecting the fluorescence using a fluorescence microscope, it can be easily determined whether or not the wild-type target gene is expressed.

  The rescue cassette of the expression vector (1-1) may have a fluorescent protein gene, a base sequence that enables polycistronic expression, and the wild-type target gene in this order (hereinafter, It may be referred to as “expression vector (1-4)”.

  FIG.2 (d) is a schematic diagram which shows the structure of an expression vector (1-4). In the figure, a case where an Ires sequence is used as a base sequence that enables polycistronic expression is shown. The expression vector (1-4) is obtained by replacing the fluorescent protein gene with the wild-type target gene in the expression vector (1-3), and is functionally the same as the expression vector (1-3). is there.

[Vector for production of expression vector (2)]
In one embodiment, the present invention is a vector for producing an expression vector used in the above-described method for producing a transgenic non-human animal, comprising a promoter, a first recombinase recognition sequence, a rescue cassette, 2 recombinase recognition sequence and the first fluorescent protein gene in this order, and the rescue cassette has a multicloning site (hereinafter referred to as “vector for production of expression vector (2-1)”). Is).

  Fig.3 (a) is a schematic diagram which shows the structure of the vector (2-1) for expression vector manufacture. The expression vector production vector (2-1) has a mutation in the target gene or a deletion of the target gene by incorporating the wild type target gene into the multiple cloning site of the rescue cassette. Is suitable for producing a vector expressing a wild-type target gene for rescue of a phenotype of a non-human animal exhibiting a different phenotype.

  The promoter, the first recombinase recognition sequence, and the second recombinase recognition sequence are the same as described above.

  As the first fluorescent protein gene, the same fluorescent protein gene as the expression vector production vector (1-1) can be used.

  Since the vector (2-1) for producing an expression vector has a multicloning site in the rescue cassette, the above vector can be produced by easily introducing a wild-type target gene.

  The expression vector production vector (2-1) may further have an enhancer, a splicing signal, a poly A addition signal, a selection marker, an origin of replication, and the like.

  When a recombinase acts on a vector obtained by introducing a wild type target gene into the expression vector production vector (2-1), the rescue cassette is excised and deleted, and the promoter and the first fluorescent protein gene are Remain. Then, the first fluorescent protein is expressed.

  As a result, fluorescence derived from the first fluorescent protein is detected at the site where the recombinase has acted.

  Therefore, for example, using a fluorescence microscope, it can be easily determined whether or not the recombinase has acted and the wild-type target gene has been deleted.

  The rescue cassette of the expression vector production vector (2-1) may have a multiple cloning site and a second fluorescent protein gene in this order (hereinafter referred to as “expression vector production vector (2-2). ) ".

  FIG. 3B is a schematic diagram showing the structure of the expression vector production vector (2-2). As the second fluorescent protein gene, the same fluorescent protein gene as the expression vector production vector (1-1) can be used, but the fluorescent protein emits fluorescence having a wavelength different from that of the first fluorescent protein gene. It is preferably a gene encoding.

  The expression vector production vector (2-2) is constructed such that when a wild-type target gene is introduced into a multicloning site, an expression vector of a fusion protein of the wild-type target gene product and a second fluorescent protein is obtained. It is preferable that The transgenic non-human animal into which the expression vector is introduced emits fluorescence derived from the second fluorescent protein at the site where the wild-type target gene is expressed.

  Further, when recombinase acts on a transgenic non-human animal into which the vector has been introduced, the rescue cassette is excised and deleted, leaving the promoter and the first fluorescent protein gene. Then, the first fluorescent protein is expressed. As a result, fluorescence derived from the first fluorescent protein is detected at the site where the recombinase has acted.

  Therefore, for example, as a result of detecting the fluorescence using a fluorescence microscope, it can be determined that the wild-type target gene is expressed at the site where the fluorescence derived from the second fluorescent protein is detected. It can be determined that the wild-type target gene has been deleted due to the action of recombinase at the site where the fluorescence derived from is detected. This determination can be easily made only by detection with a fluorescence microscope.

  It should be noted that a sequence that reliably terminates transcription is preferably present on the 3 'side of the second fluorescent protein gene. Otherwise, the first fluorescent protein may be expressed together with the second fluorescent protein. Examples of the sequence that surely terminates transcription include a poly A addition signal.

  The rescue cassette of the expression vector production vector (2-1) may have a multicloning site, a base sequence enabling polycistronic expression, and a second fluorescent protein gene in this order. (Hereafter, it may be referred to as “expression vector production vector (2-3)”.)

  FIG.3 (c) is a schematic diagram which shows the structure of the vector (2-3) for expression vector manufacture. In the figure, a case where an Ires sequence is used as a base sequence that enables polycistronic expression is shown. Expression vector production vector (2-3) is such that when a wild-type target gene is introduced into a multicloning site, an expression vector that expresses both the wild-type target gene and the second fluorescent protein gene is obtained. It is preferable that it is constructed.

  In a transgenic non-human animal into which the expression vector has been introduced, for example, as a result of detecting fluorescence using a fluorescence microscope, a wild-type target gene is expressed at a site where fluorescence derived from the second fluorescent protein is detected. It can be determined that the wild-type target gene has been deleted due to the action of recombinase at the site where the fluorescence derived from the first fluorescent protein is detected. This determination can be easily made only by detection with a fluorescence microscope.

  The rescue cassette of the expression vector production vector (2-1) may have a second fluorescent protein gene, a base sequence enabling polycistronic expression, and a multicloning site in this order. (Hereinafter, it may be referred to as “vector for producing expression vector (2-4)”.)

  FIG. 3 (d) is a schematic diagram showing the structure of a vector (2-4) for producing an expression vector. In the figure, a case where an Ires sequence is used as a base sequence that enables polycistronic expression is shown. The expression vector production vector (2-4) is obtained by replacing the second fluorescent protein gene and the multicloning site in the expression vector production vector (2-3), and functionally for producing the expression vector. It is the same as the vector (2-3).

[Expression vector (2)]
In one embodiment, the present invention comprises a promoter, a first recombinase recognition sequence, a rescue cassette, a second recombinase recognition sequence, and a first fluorescent protein gene in this order, wherein the rescue cassette An expression vector (hereinafter sometimes referred to as “expression vector (2-1)”) having a wild-type target gene is provided.

  Fig.4 (a) is a schematic diagram which shows the structure of an expression vector (2-1). The expression vector (2-1) can be suitably used for the production of the above-described transgenic non-human animal.

  The expression vector (2-1) can be obtained by introducing a wild-type target gene into the multiple cloning site of the rescue cassette of the above-described expression vector production vector (2-1).

  When a recombinase acts on a transgenic non-human animal into which an expression vector (2-1) has been introduced, the rescue cassette is excised and deleted, leaving the promoter and the first fluorescent protein gene. Then, the first fluorescent protein is expressed. As a result, fluorescence derived from the first fluorescent protein is detected at the site where the recombinase has acted.

  Therefore, for example, using a fluorescence microscope, it can be easily determined whether or not the recombinase has acted and the wild-type target gene has been deleted.

  The rescue cassette of the expression vector (2-1) may have a gene encoding a fusion protein of the protein encoded by the wild-type target gene and the second fluorescent protein (hereinafter referred to as “expression vector”). (2-2) ".

  FIG. 4B is a schematic diagram showing the structure of the expression vector (2-2). The expression vector (2-2) can be obtained by introducing a wild-type target gene into the multiple cloning site of the rescue cassette of the above-described expression vector production vector (2-2).

  The transgenic non-human animal into which the expression vector (2-2) has been introduced emits fluorescence derived from the second fluorescent protein at the site where the wild-type target gene is expressed.

  Furthermore, when a recombinase acts on a transgenic non-human animal into which the vector (2-2) has been introduced, the rescue cassette is excised and deleted, leaving the promoter and the first fluorescent protein gene. Then, the first fluorescent protein is expressed. As a result, fluorescence derived from the first fluorescent protein is detected at the site where the recombinase has acted.

  Therefore, for example, as a result of detecting the fluorescence using a fluorescence microscope, it can be determined that the wild-type target gene is expressed at the site where the fluorescence derived from the second fluorescent protein is detected. It can be determined that the wild-type target gene has been deleted due to the action of recombinase at the site where the fluorescence derived from is detected. This determination can be easily made only by detection with a fluorescence microscope.

  The rescue cassette of the expression vector (2-1) may have the wild-type target gene, a base sequence that enables polycistronic expression, and a second fluorescent protein gene in this order. (Hereinafter, it may be referred to as “expression vector (2-3)”.)

  FIG. 4C is a schematic diagram showing the structure of the expression vector (2-3). In the transgenic non-human animal into which the expression vector (2-3) has been introduced, the wild-type target gene is detected at the site where the fluorescence derived from the second fluorescent protein is detected as a result of detecting fluorescence using, for example, a fluorescence microscope. Can be determined, and at the site where fluorescence derived from the first fluorescent protein is detected, it can be determined that the recombinase acts to delete the wild-type target gene. This determination can be easily made only by detection with a fluorescence microscope.

  The rescue cassette of the expression vector (2-1) may have the second fluorescent protein gene, a base sequence that enables polycistronic expression, and the wild-type target gene in this order. (Hereinafter, it may be referred to as “expression vector (2-4)”.)

  FIG.4 (d) is a schematic diagram which shows the structure of an expression vector (2-4). In the figure, a case where an Ires sequence is used as a base sequence that enables polycistronic expression is shown. The expression vector (2-4) is obtained by replacing the second fluorescent protein gene and the multicloning site in the expression vector production vector (2-3), and is functionally an expression vector (2-3). It is the same.

  Next, although an experiment example is shown and this invention is demonstrated further in detail, this invention is not limited to the following experiment examples.

[Experimental example]
A group in which an expression vector having a structure of “loxP sequence” — “EF1α promoter” — “gene encoding a fusion protein of Inv gene and green fluorescent protein (GFP)” — “loxP sequence” is introduced into ES cells (control) Group) was prepared. In addition, a group (test group) in which an expression vector of Cre recombinase was further introduced into the ES cells was prepared. The Inv gene is a gene having an important function in the formation of the left and right axes and kidney formation.

  Each of the above cells was immunostained with an anti-GFP antibody and observed with a fluorescence microscope. FIG. 5 shows a fluorescence micrograph. FIG. 5 (a) is a photograph of the cells in the control group, and FIG. 5 (b) is a photograph of the cells in the test group.

  As a result, no GFP-expressing cells were observed in the test group cells, whereas many GFP-expressing cells were observed in the control group cells. This result shows that in the cells of the test group, the “gene encoding the fusion protein of Inv gene and green fluorescent protein (GFP)” was deleted due to gene recombination with Cre recombinase. Indicates functioning.

  According to the present invention, there is provided a method for producing a non-human animal that can more easily analyze a time-specific or tissue-specific gene function and can be applied to an animal in which ES cells are not established. be able to. Moreover, the non-human animal manufactured by said manufacturing method, the expression vector used for said manufacturing method, and the vector for the said expression vector manufacture can be provided.

Claims (2)

An expression vector having a first recombinase recognition sequence, a wild-type target gene, and a second recombinase recognition sequence in this order,
A non-human animal having a mutation in the target gene and showing a phenotype different from the wild type, and a fertilized egg of a non-human animal in which ES cells are not established, or the target gene is deleted and the wild type Introducing a fertilized egg of a non-human animal having a phenotype different from that of a non-human animal in which ES cells have not been established;
Culturing the fertilized egg to obtain a transgenic non-human animal;
A method for producing a transgenic non-human animal in which the phenotype is rescued. A transgenic non-human animal is produced by the production method according to claim 1, and then the recombinase is caused to act on the transgenic non-human animal in a time-specific or tissue-specific manner, and the introduced wild-type target gene is introduced. A method for producing a genetically modified non-human animal, wherein the target gene is mutated or deleted in a time-specific or tissue-specific manner.