Method for producing transgenic pigs, somatic cell cloning material, transgenic pigs, established somatic cells, and tissues or organs

Abstract

To provide a method for producing a transgenic pig into which a gene group for expressing specific functional proteins in a tissue or an organ is introduced selectively into this tissue or organ, somatic cell cloning materials used therefor, a transgenic pig, somatic cells established therefrom, and the like.SOLUTION: A method for producing a transgenic pig comprises a step for carrying out gene introduction by intracytoplasmic-sperm injection of liquid into an ovum, the liquid comprising a vector (1) and a genetically modified cloned pig sperm obtained by somatic-cell cloning of a genetically modified pig cell obtained by inserting, into a safe harbor region, a gene cassette containing a gene encoding a functional protein and a construct that allows expression of the functional protein using a recombinant enzyme, wherein the vector (1) contains a promoter for expression in a specific tissue or organ and a gene encoding the recombinant enzyme that exists downstream of the promoter and is under the control of the promoter for the expression in the specific tissue or the organ.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for producing a transgenic pig, a somatic cell cloning material used therein, a transgenic pig, somatic cells established therefrom, and tissues or organs extracted therefrom. [Background technology]

[0002] There is a need to develop transgenic pigs that can express specific functional proteins in tissue- or organ-selective, conditional, and even time-selective, tissues or organs. Summary of the Invention [Problem to be solved by the invention]

[0003] Since breeding or mating of pigs is complicated compared to rodents such as mice, the inventors initially attempted to produce transgenic pigs by introducing a vector containing both a control gene cassette for conditional gene expression and a functional gene cassette containing a gene encoding a functional protein into pigs. However, the inventors have found that when a vector containing both a control gene cassette for conditional gene expression and a functional gene cassette containing a gene encoding a functional protein is introduced into a pig, it is difficult to produce a transgenic pig into which a group of genes for conditionally expressing the functional protein in a specific tissue or organ has been introduced (Comparative Example described below).

[0004] The present invention has been made in consideration of the problems with the prior art as described above, and an object of the present invention is to provide a method for producing a transgenic pig into which a group of genes for expressing a specific functional protein in a tissue or organ selectively is introduced, and the somatic cell cloning material, transgenic pig, somatic cells established from the transgenic pig, and tissues or organs extracted from the transgenic pig. [Means for solving the problem]

[0005] As a result of intensive research into the above-mentioned problems, the present inventors have found that by dividing the gene transfer into two steps, that is, by gene transfer using a functional gene cassette containing a gene encoding a functional protein and gene transfer using a vector containing a control gene cassette for conditional gene expression, it is possible to produce a transgenic pig into which a group of genes for conditionally expressing a functional protein in a tissue or organ has been introduced. In other words, it has been found that by constructing a genetically modified cloned pig into which a functional gene cassette containing a gene encoding a functional protein has been introduced, and then further gene-transforming the constructed genetically modified cloned pig with a vector containing a control gene cassette, it is possible to produce a transgenic pig into which a group of genes for conditionally expressing a functional protein in a tissue or organ has been introduced. The present invention has been completed based on the above findings. That is, the present invention is as follows.

[0006] <1> Sperm of a genetically modified cloned pig obtained by somatic cloning of a genetically modified pig cell obtained by introducing a gene cassette containing a gene encoding a functional protein and a construct capable of expressing the functional protein by a recombinase into a safe harbor region; A method for producing a transgenic pig, comprising the step of introducing a gene into an egg by insemination with a liquid containing the vector (1), The vector (1), The production method includes a promoter that is expressed in a specific tissue or organ, and a gene encoding the recombinant enzyme that is located downstream of and under the control of the promoter that is expressed in the specific tissue or organ. <2> the safe harbor area is a ROSA area; <1> The method described above. <3> The vector (1) is a plasmid vector or an artificial chromosome vector. <1> The method described above. <4> the above <1> ~ <3> 4. A somatic cell established from a transgenic pig produced by the method according to claim 1.

[0007] <5> A somatic cell cloning source comprising a genetically modified porcine cell comprising a gene encoding a functional protein, the gene cassette comprising a construct capable of expressing the functional protein by a recombinase in a safe harbor region, A somatic cell cloning material to provide a material for gene transfer by ICSI in the presence of the following vector (1). (1) A vector comprising a promoter that is expressed in a specific tissue or organ, and a gene encoding the recombinase that is downstream of and under the control of the promoter that is expressed in the specific tissue or organ. <6> A gene encoding the recombinase is contained in the downstream of a promoter that is expressed in a specific tissue or organ and is under the control of the promoter that is expressed in the specific tissue or organ, A transgenic pig comprising a gene encoding a functional protein, the transgenic pig comprising a gene cassette in a safe harbor region, the gene cassette comprising a construct that enables expression of the functional protein by the recombinant enzyme. <7> the above <6> 2. A somatic cell line established from the transgenic pig described in . <8> the above <6> A tissue or organ isolated from the transgenic pig described in 1. Effect of the Invention

[0008] According to the present invention, it is possible to provide a method for producing a transgenic pig into which a group of genes has been introduced for conditionally expressing a specific functional protein in a tissue or organ selectively (preferably in a tissue or organ-specific manner), as well as somatic cell cloning materials used therein, a transgenic pig, somatic cells established from the pig, and tissues or organs extracted from the pig. [Brief description of the drawings]

[0009] [Figure 1]FIG. 1 is a schematic diagram showing a gene cassette containing the loxP-Venus gene constructed in Reference Example 1. [Diagram 2] FIG. 2 is a schematic diagram showing the CreERT2 expression vector constructed in Reference Example 2. [Diagram 3] FIG. 3 is a schematic diagram showing a gene cassette containing the loxP-DTA gene constructed in Reference Example 3. [Figure 4] FIG. 4 is an electrophoretogram showing the results of confirming the knock-in of the above gene cassette into the ROSA region. [Diagram 5] FIG. 5 shows the results of confirming the induction of cell death by tamoxifen in genetically modified porcine cells in which a gene cassette containing the loxP-DTA gene was introduced into the porcine ROSA region. [Figure 6] FIG. 6 shows electrophoretograms of the sex determination results. [Figure 7] FIG. 7 shows electrophoretic patterns indicating the results of gene transfer evaluation. [Figure 8] FIG. 8 is an electrophoretogram showing the results of confirming CreERT2 expression in fetal metanephroi. [Figure 9] FIG. 9 shows the results of transgene copy number determination in individuals (diploid genome). [Figure 10] FIG. 10 is a schematic diagram showing an all-in-one vector 1 containing both the mSix2 promoter and the CreERT2 and loxP-DTA genes as a comparative example. [Figure 11] FIG. 11 is a schematic diagram showing an all-in-one vector 2 containing both the mSix2 promoter and the ERT2CreERT2 and loxP-DTA genes as a comparative example. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The following describes in detail the embodiments of the present invention. However, the present invention is not limited to the following embodiments and can be implemented with appropriate modifications within the scope of the object of the present invention.

[0011] <Production method for transgenic pigs> The first aspect of the present invention relates to a method for producing a pig that is capable of expressing a functional protein by a recombinant enzyme, comprising: introducing a gene cassette containing a construct into a safe harbor region; and subjecting the resulting gene cassette to somatic cloning of a genetically modified pig cell. The present invention relates to a method for producing a transgenic pig, the method including the step of introducing a gene into an egg by microinjection with a liquid containing the vector (1). (1) A vector comprising a promoter that is expressed in a specific tissue or organ, and a gene encoding the recombinase that is located downstream of and under the control of the promoter that is expressed in a specific tissue or organ.

[0012] The transgenic pig produced by the production method according to the first aspect has undergone gene transfer (first gene transfer) of the gene cassette into the safe harbor region and second gene transfer by the vector (1) via the in vitro fertilization, and can also be called a double transgenic pig (double genetically modified pig). In the present invention, the transgenic pig is preferably a transgenic pig that can conditionally express a specific functional protein in a tissue or organ selectively.

[0013] The transgenic pig produced by the production method according to the first aspect comprises a gene encoding a functional protein, and a gene cassette including a construct that enables the functional protein to be expressed by a recombinase. In addition, the transgenic pig produced by the production method according to the first aspect comprises a promoter that is expressed in a specific tissue or organ, and a gene encoding the recombinant enzyme that is located downstream of the promoter and under its control. As a result, by coexisting the recombinant enzyme with the construct (preferably within the cell, more preferably within the nucleus), the functional protein can be conditionally expressed (preferably, dependent on the expression of the recombinant enzyme). In other words, in the production method according to the first aspect, by microinjecting an egg with a liquid containing a vector (1) containing a gene encoding the recombinant enzyme, a transgenic pig can be produced that is capable of conditionally (preferably, dependent on the expression of the recombinant enzyme) expressing the functional protein. As a result, the transgenic pig produced by the production method of the first embodiment can express the functional protein in a tissue or organ selectively (preferably, tissue or organ specifically).

[0014] In pigs, a large amount (high copy number) of control gene cassettes is required for conditional gene expression, while a small amount (low copy number) of functional gene cassettes containing genes encoding functional proteins is sufficient. When sufficient gene transfer (transfection) is achieved to perform conditional expression by the control gene cassette, the copy number of the functional gene cassette is too large, and the quantitative balance between the control gene cassette and the functional gene cassette is not adequate for pigs. The present inventors speculate that this is a problem of the prior art that makes it difficult to produce the transgenic pigs. The present inventors also speculate that the occurrence of non-specific expression (leakage) of the functional gene cassette is also a reason for the problem of the prior art that makes it difficult to produce the transgenic pigs.

[0015] More specifically, for example, when the integrated copy number of an all-in-one vector (see the comparative example described below) containing both the control gene cassette and the functional gene cassette is low, the expression level of the gene that expresses a recombinase in a tissue-specific manner becomes insufficient, and as a result, the expression of the gene encoding the functional protein becomes insufficient. On the other hand, if the integrated copy number of an all-in-one vector containing both the control gene cassette and the functional gene cassette is high, the expression of the gene encoding the functional protein will be excessive due to leakage, which will affect the efficiency of producing transgenic pigs.

[0016] In the transgenic pig produced by the production method of the first aspect and the transgenic pig of the third aspect described below, the copy number of the gene encoding the functional protein is not particularly limited, but may be, for example, one or more copies per pig individual (diploid genome), preferably two or more copies, and more preferably four or more copies. There is no particular upper limit on the number of copies of the gene encoding the functional protein, but examples include 200 copies or less, 100 copies or less, or 550 copies or less per pig individual (diploid genome).

[0017] In the transgenic pig produced by the production method of the first aspect and the transgenic pig produced by the transgenic pig of the third aspect described below, the copy number of the gene encoding the recombinant enzyme is not particularly limited, but may be, for example, 1 or more copies per pig individual (diploid genome), with 2 or more copies being preferred, 4 or more copies being more preferred, 7 or more copies being even more preferred, 15 or more copies being particularly preferred, and 30 or more copies being particularly preferred. There is no particular upper limit to the copy number of the recombinase, but examples include 500 copies or less, 300 copies or less, or 100 copies or less per pig individual (diploid genome).

[0018] In the transgenic pig produced by the production method of the first aspect and the transgenic pig of the third aspect described below, the ratio of the copy number of the gene encoding the functional protein to the copy number of the gene encoding the recombinant enzyme is, for example, 1 / 2 to 1 / 300, preferably 2 / 3 to 2 / 100, more preferably 3 / 5 to 3 / 50, and even more preferably 4 / 6 to 4 / 20.

[0019] In the first aspect, the construct is not particularly limited as long as it contains a gene encoding the functional protein and the functional protein can be expressed by a recombinant enzyme, and examples of the construct include nucleic acid constructs such as DNA constructs, cDNA constructs, and RNA constructs (preferably gene constructs). In the first aspect, the construct preferably comprises a specific sequence that is selectively (preferably specifically) recognized by the recombinase, and more preferably comprises at least one pair of specific sequences that are selectively (preferably specifically) recognized by the recombinase. As a result, the recombinase selectively recognizes at least one pair of specific sequences (preferably at least one pair of specific sequences), thereby enabling conditional (preferably dependent on expression of the recombinase)) recombination to occur.

[0020] Combinations of the above-mentioned recombinase and a specific sequence (preferably at least one pair of specific sequences) selectively recognized thereby include the following combinations (a) to (d), of which the following combination (a) of Cre recombinase and at least one pair of Lox sequences is preferred. (a) a combination of Cre recombinase and at least one pair of Lox sequences; (b) A combination of the recombinase FLP derived from Saccharomyces cerevisiae and the following FRT sequence (Broach, JR, Guarascio, VR, & Jayaram, M. (1982). Recombination within the yeast plasmid 2mu circle is site-specific. Cell, 29(1), 227-34.) FRT sequence: 5'-GAAGTTCCTATTCTCTAGAAAGTATAGGAACTTC-3' (SEQ ID NO: 7) (c) A combination of the recombinant enzyme R and RS sequences derived from soy sauce yeast (Zygosaccharomyces rouxii) (Araki, H., Jearnpipatkul, A., Tatsumi, H., Sakurai, T., Ushio, K., Muta, T., & Oshima, Y. (1985). Molecular and functional organization of yeast plasmid pSR1. Journal of molecular biology, 182(2), 191-203.) (d) Combination of the recombinase Gin and gix sequences derived from bacteriophage Mu (Maeser, S., & Kahmann, R. (1991). The Gin recombinase of phage Mu can catalyse site-specific recombination in plant protoplasts. Molecular & general genetics: MGG, 230(1-2), 170-6.)

[0021] The specific sequence is preferably the Lox sequence, and among the Lox sequences, a representative example is the 34-base loxP sequence derived from bacteriophage P1, which allows the construction of a so-called Cre-loxP system. An example of a pair of loxP sequences is shown below. 5'-ATAACTTCGTATAGCATACATTATACGAAGTTAT-3' (SEQ ID NO: 1) As shown above, the 13 bases at both ends of the Cre binding site are symmetrical, while the central 8 bases are asymmetrical. loxP has a directionality, and if a pair of Lox sequences are arranged in the same direction (same orientation), the sequence between them is excised as a circular DNA by Cre. The excised circular DNA rarely returns to its original state, and the excision is essentially irreversible. On the other hand, if a pair of Lox sequences are arranged in the opposite direction (opposite orientation), the sequence between them is inverted (the orientation is swapped). The above inversion is said to be reversible.

[0022] In the first embodiment, the at least one pair of Lox sequences preferably comprises 1 to 4 pairs of Lox sequences, more preferably 1 to 3 pairs of Lox sequences, and even more preferably 1 or 2 pairs of Lox sequences. Representative examples of at least one pair of Lox sequences include not only the loxP sequence described above, but also lox511 sequences, lox2272 sequences, loxFAS sequences, loxRE sequences, loxLE sequences, etc., and two or more pairs of these may be used in combination.

[0023] Examples of the lox511 sequence, the lox2272 sequence, the loxFAS sequence, the loxRE sequence, and the loxLE sequence are shown below. In the sequences below, the lowercase letters indicate bases that differ from the loxP sequence. lox511: 5'-ATAACTTCGTATAGtATACATTATACGAAGTTAT-3' (SEQ ID NO: 2) lox2272: 5'-ATAACTTCGTATAGgATACtTTATACGAAGTTAT-3' (SEQ ID NO: 3) loxFAS: 5'-ATAACTTCGTATAtacctttcTATACGAAGTTAT-3' (SEQ ID NO: 4) lox RE: 5'-ATAACTTCGTATAGCATACATTATACGAAcggta-3' (SEQ ID NO: 5) lox LE: 5'-taccgTTCGTATAGCATACATTATACGAAGTTAT-3' (SEQ ID NO: 6)

[0024] It is known that specific recombination occurs between loxP-loxP, lox511-lox511, and lox2272-lox2272, respectively, whereas no recombination occurs between loxP-lox511 or loxP-lox2272. By utilizing this specific recombination property, two or more pairs of Lox sequences (placed in the opposite direction between each pair of sequences) can be used to place a target gene (a gene encoding a functional protein) in the opposite direction (inactive type), and then it can be made into a FLEx (Flip-excision) switch that irreversibly recombines with Cre to activate it by exon flipping (Experimental Medicine, November 2014, Vol. 32, No. 18). This overcomes the reversibility of inversion when a pair of Lox sequences is placed in the opposite direction (inverted).

[0025] When used as a FLEx switch, examples of the combination of two pairs of Lox sequences include a combination of one pair of loxP and one pair of lox2272, and a combination of one pair of loxP and one pair of lox511.

[0026] In addition to the FLEx switch, an example of a configuration in which a pair of Lox sequences is used is described below. In the above gene cassette, the gene of interest (a gene encoding a functional protein) can be arranged in an active orientation, and a STOP sequence flanked (Floxed) by a pair of Lox sequences arranged in the same direction (same orientation) can be arranged upstream of the gene of interest. Since a pair of Lox sequences are arranged in the same direction (same orientation), the STOP sequence is essentially irreversibly excised by Cre, allowing the expression of the target gene (a gene encoding a functional protein). The STOP sequence is not particularly limited as long as it is a sequence for terminating transcription, but examples thereof include the SV40 polyA signal sequence, and a sequence in which three SV40 polyA signal cassettes are linked is preferred.

[0027] From the viewpoint of preventing non-specific recombination (leakage), a Pause sequence may be included in addition to the STOP sequence as a Floxed sequence between a pair of Lox sequences arranged in the same direction (same orientation), and it is preferable that a Pause sequence is included downstream of the STOP sequence. The pause sequence is not particularly limited, but examples thereof include the human α2 globin gene RNA polymerase II transcriptional pause signal sequence.

[0028] In the production method according to the first aspect, the recombinant enzyme is preferably a fusion protein with a nuclear transport protein, i.e., the gene encoding the recombinant enzyme is preferably fused directly or indirectly with a gene encoding the nuclear transport protein. The nuclear transport protein is not particularly limited as long as it can be transported into the nucleus, but examples thereof include proteins having a nuclear transport signal, and specifically, the estrogen receptor ER is preferred. It is more preferable that the recombinase is Cre recombinase, and the gene encoding the recombinase is a gene encoding a fusion protein containing the Cre recombinase and the estrogen receptor ER. The above-mentioned Cre recombinase is known as an enzyme (derived from bacteriophage P1) that recognizes specific base sequences (for example, a pair of two Lox sequences) and causes DNA homologous recombination. The estrogen receptor ER is known to have a nuclear localization signal. When the fusion protein is a fusion protein containing Cre recombinase and the estrogen receptor ER, a liquid containing the vector (1) is microinjected into an egg, whereby the Cre recombinase portion of the fusion protein recognizes at least one pair of Lox sequences, thereby producing a transgenic pig that undergoes conditional (preferably Cre expression-dependent) recombination.

[0029] In the production method according to the first aspect, it is more preferable that the estrogen receptor ER is ERT2 and the fusion protein is CreERT2 or ERT2CreERT2. Tamoxifen compounds (tamoxifen, 4-hydroxytamoxifen (4-OHT), etc.) can bind to ER present in the cytoplasm and translocate into the nucleus. ERT2 is known to be a protein that has been modified to bind ER only to tamoxifen compounds and lacks a nuclear localization signal (with estrogen). Both CreERT2 and ERT2CreERT2 are fusion proteins of the above-mentioned ERT2 and Cre, and can translocate into the nucleus in the presence of a tamoxifen compound and cause recombination by Cre. (Feil R, Brocard J, Mascrez B, LeMeur M, Metzger D and Chambon P (1996): Ligand-activated site-specific recombination in mice. Proc Natl Acad Sci USA, 93: 10887-10890. 1996. Feil R, Wagner J, Metzger D and Chambon P (1997): Regulation of Cre recombinase activity by mutated estrogen receptor ligand-binding domains. Biochemical and Biophysical Research Communications, 237: 752-727. 1997.) In a first embodiment, CreERT2 or ERT2CreERT2 can be used to induce conditional (eg, time-specific) recombination by tamoxifen compounds. In the first and second aspects, said estrogen receptor ER is preferably ERT2 and said fusion protein is preferably CreERT2 or ERT2CreERT2.

[0030] (A genetically modified porcine cell containing a gene encoding the functional protein and a gene cassette containing a construct that enables the functional protein to be expressed by a recombinase in a safe harbor region) The present invention is a somatic cell cloning material comprising a genetically modified pig cell containing a gene encoding the functional protein and containing, in a safe harbor region, a gene cassette containing a construct that enables the functional protein to be expressed by a recombinase, and the somatic cell cloning material is also an invention relating to a somatic cell cloning material for providing a material for gene introduction by intracytoplasmic sperm injection in the presence of the vector (1). A second aspect of the present invention is the somatic cell cloning material.

[0031] In the first and second aspects, the genetically modified porcine cells are preferably established (e.g., established strains or cloned) genetically modified porcine cells, from the viewpoints of stable culture and high cell homogeneity. Furthermore, by establishing this system, it is possible to omit the various steps required prior to establishment, such as introducing a gene cassette into a safe harbor region, and to produce transgenic pigs into which a group of genes for conditionally expressing the above-mentioned functional protein in a specific tissue or organ can be introduced in a single step (e.g., essentially in one step) of the above-mentioned in vitro fertilization, which makes this system excellent for industrial mass production of transgenic pigs.

[0032] The above-mentioned genetically modified porcine cells are not particularly limited as long as they are porcine cells having a safe harbor region (preferably in the genome), but examples thereof include fibroblasts, preadipocytes, embryo-derived pluripotent cells, somatic stem cells, induced pluripotent stem cells, etc., with fibroblasts being preferred and fetal fibroblasts being more preferred.

[0033] The safe harbor region into which the gene cassette is introduced in the first and second aspects refers to a region on the genome into which a transgene can be safely inserted, and examples of such regions include non-coding regions on the genome into which a transgene can be safely inserted. As the safe harbor region, the porcine ROSA region or the H11 region is preferable, and the porcine ROSA region is more preferable. The porcine ROSA region is not particularly limited as long as it is a ROSA region, but the ROSA region located between exons 1 and 2 of the 13th porcine chromosome is preferred.

[0034] In the first and second aspects, the functional protein can be expressed by the action of the recombinant enzyme in cells throughout the body of the pig (systemic expression), or in cells of a specific tissue or organ (localized expression), but it is preferable that the functional protein be expressed at least in the "specific tissue or organ" defined in the vector (1). Compared to rodents such as mice, for example, breeding or mating of pigs is complicated. From the viewpoint that various gene-transduced pig variations capable of conditionally expressing the functional protein in specific tissues or organs can be produced in a single step (e.g., substantially one process) of intracytoplasmic sperm injection by preparing various vectors (1) containing promoters expressed in various tissues or organs and performing intracytoplasmic sperm injection in the presence of various vectors (1), it is preferable that the functional protein be expressible (systemically expressible) in cells throughout the body of the pig individual. When the gene cassette containing the construct is introduced into a safe harbor region in the genome, it can be expressed in cells throughout the body of the pig (systemic expressibility).

[0035] In the gene cassettes of the first and second aspects, the functional protein is not particularly limited as long as it is a protein that exerts any function, and examples thereof include proteins that exert any function, such as a light-emitting (e.g., fluorescence, UV light) function, a labeling function (radioactive label, any tag), a gene expression regulating function, a gene recognition function, and a function that is lethal to a cell (e.g., acting on the cytoplasmic membrane to destroy it). More specifically, examples thereof include fluorescent proteins (e.g., modified GFPs such as Green Fluorescent Protein (GFP) and Venus), metabolic enzymes (e.g., thymidine kinase, drug metabolizing enzymes (CYP)), cell proliferation-related factors (e.g., apoptosis regulator BAX, fibroblast growth factor FGF, etc.), growth-related gene proteins (e.g., growth hormone receptor, insulin-like growth factor receptor, etc.), cytotoxins, suicide gene proteins (e.g., iCaspase9, etc.), and the like. Fluorescent proteins are generally known to be cytotoxic. The cytotoxin is not particularly limited as long as it has a lethal effect on cells (for example, by acting on the cytoplasmic membrane and destroying it), and is preferably a toxic substance with a high molecular weight, more preferably a toxic protein. Specific examples of the cytotoxin include diphtheria toxin, θ toxin, streptolysin S, α, δ toxin, leukocidin, etc., and diphtheria toxin is preferred, and diphtheria toxin A is more preferred. Diphtheria toxin is a bacterial toxin produced by the diphtheria bacterium, and consists of two domains, A chain and B chain. The A chain acts as the toxin itself, while the B chain binds to a receptor on the cell surface and takes the A chain into the cell.

[0036] In the gene cassettes of the first and second aspects, the gene encoding the cytotoxin includes genes encoding the above-mentioned cytotoxins, preferably a gene encoding diphtheria toxin, and more preferably a gene encoding diphtheria toxin A (hereinafter also simply referred to as "DTA").

[0037] In the gene cassettes of the first and second aspects, in order to allow the gene cassette to accept transcription by the promoter of the safe harbor region or a promoter located upstream of the safe harbor region, the gene cassette may or may not further include a splicing acceptor sequence (splice acceptor sequence) including AG or the like, but it is preferable that the gene cassette further includes a splice acceptor sequence, it is more preferable that the gene cassette further includes a splice acceptor sequence upstream of a "construct including a gene encoding a functional protein and enabling the functional protein to be expressed by a recombinase", and it is even more preferable that the gene cassette further includes a splice acceptor sequence upstream of a "specific sequence and a gene encoding a functional protein".

[0038] In the gene cassette containing the construct in the first and second aspects, in order to control the expression of the functional protein, an arbitrary promoter may or may not be further contained upstream of a gene encoding the functional protein (preferably a specific sequence and a gene encoding the functional protein). The arbitrary promoter may be a promoter that functions in any cell (preferably various types of cells), and preferably includes a CAG (cytomagalovirus immediate early enhancer-chiken β-actin hybrid), CMV, RSV promoter, etc., and more preferably a CAG promoter.

[0039] In the gene cassettes containing the above constructs in the first and second aspects, they may or may not further contain a drug selection sequence for selecting genetically modified pig cells (gene knock-in pig cells). If they further contain such a sequence, it is preferable that they are contained downstream of a gene encoding a functional protein. The above-mentioned drug selection sequence is not particularly limited, but examples thereof include a gene cassette consisting of SV40 promoter, puromycin N-acetyltransferase, and pA (polyA) of herpes simplex virus thymidine kinase (HSV-TK).

[0040] In the first and second aspects, a genome editing method can be used as a method for introducing the gene cassette into the above-mentioned safe harbor region (first gene modification method). It is preferable to introduce the above-mentioned gene cassette and genome editing tool into a cell (preferably into the nucleus) by any transgene introduction technique such as lipofection, electroporation, calcium phosphate, etc., and then introduce them into the above-mentioned safe harbor region by genome editing.

[0041] Introduction by genome editing methods includes introduction using a genome editing tool containing an artificial nuclease for genome editing such as CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas nuclease, and may also be introduction using a genome editing tool containing an artificial restriction enzyme (artificial nuclease) using Transcription Activator-Like Effector Nuclease (TALEN) and zinc finger nuclease (ZFN).

[0042] The CRISPR / Cas nuclease comprises a guide RNA and a Cas nuclease (preferably Cas9). At least a partial sequence within the safe harbor region that is targeted includes an oligonucleotide contained within the safe harbor region.

[0043] A gene cassette containing the above construct can be introduced by transfecting a guide RNA or a DNA encoding a guide RNA specific to at least a partial sequence in the above safe harbor region, and a composition containing a nucleic acid encoding a Cas nuclease or a Cas nuclease into a eukaryotic cell or eukaryotic organism containing the above safe harbor region. Nucleic acid encoding Cas nuclease or Cas nuclease and guide RNA or DNA encoding guide RNA can be introduced into cells by various methods known in the art, such as, but not limited to, microinjection, electroporation, DEAE-dextran treatment, lipofection, nanoparticle-mediated transfection, protein transduction domain-mediated transduction, virus-mediated gene delivery, and PEG-mediated transfection into protoplasts. Nucleic acid encoding Cas nuclease or Cas nuclease and guide RNA can also be introduced into an organism by various methods known in the art for administering genes or proteins, such as injection. Nucleic acid encoding Cas nuclease or Cas protein can be introduced into cells in the form of a complex with guide RNA or separately. Cas nuclease fused with a protein transduction domain such as Tat can also be efficiently delivered into cells. Preferably, the eukaryotic cell or organism is co-transfected or sequentially transfected with the Cas9 nuclease and the guide RNA. Sequential transfection can be performed by first transfection with a nucleic acid encoding a Cas nuclease, followed by a second transfection with a naked guide RNA, preferably, but not limited to, the second transfection 3, 6, 12, 18, 24 hours later.

[0044] The expression of the guide RNA may be performed using a guide RNA expression unit. The guide RNA expression unit is preferably a transcription unit of the CRISPR-Cas9 system including a target sequence (at least a partial sequence in the above-mentioned safe harbor region) and a guide RNA, and preferably has a promoter region for expressing the guide RNA (a promoter of RNA polymerase III (e.g., a promoter selected from U6 promoter and H1 promoter)), a target sequence (at least a partial sequence in the above-mentioned safe harbor region) and a guide RNA, and more preferably, the promoter, a sequence complementary to the target sequence (at least a partial sequence in the above-mentioned safe harbor region) and the guide RNA are seamlessly linked. In order to prevent off-target, the CRISPR / Cas nuclease can also use a Cas9 mutant that cuts only one strand of double-stranded DNA as a nickase. An example of a single-strand cleavage type Cas9 mutant is Cas9(D10A). For example, when a single-strand cleavage type Cas9 mutant is used in combination with a guide RNA having a target sequence complementary to one strand of a target DNA and a guide RNA having a target sequence complementary to the other strand in close proximity thereto, one strand is cleaved with a specificity of 20 bases and the other strand is cleaved with a specificity of another 20 bases, resulting in a total cleavage of DNA with a specificity of 40 bases, making it possible to significantly improve the specificity of the target.

[0045] Genetically modified pig cells (gene knock-in pig cells) obtained by introducing the gene cassettes of the first and second aspects into the above-mentioned safe harbor region can be selected and established by any method such as limiting dilution of less than one cell per well, genetic analysis, drug resistance, etc.

[0046] The following documents (I) to (III) describe the establishment of genetically modified pig cells by genome editing and the production of genetically modified cloned pigs by somatic cloning of the established genetically modified pig cells. (i)Masahito Watanabe, Kazuhiro Umeyama, Kazuaki Nakano, Hitomi Matsunari, Toru Fukuda, Kei Matsumoto, Susumu Tajiri, Shuichiro Yamanaka, Koki Hasegawa, Kazutoshi Okamoto, Ayuko Uchikura, Shuko Takayanagi, Masaki Nagaya, Takashi Yokoo, Hiromitsu Nakauchi and Hiroshi Nagashima (2022) Generation of heterozygous PKD1 mutant pigs exhibiting earlyonset renal cyst formation. Laboratory Investigation, 102:560-569; https: / / doi.org / 10.1038 / s41374-021-00717-z (b)Hitomi Matsunari, Michiyo Honda, Masahito Watanabe, Satsuki Fukushima, Kouta Suzuki, Shigeru Miyagawa, Kazuaki Nakano, Kazuhiro Umeyama, Ayuko Uchikura, Kazutoshi Okamoto,Masaki Nagaya, Teruhiko Toyo-oka, Yoshiki Sawa, Hiroshi Nagashima (2020) Pigs with δ-sarcoglycan deficiency exhibit traits of genetic cardiomyopathy. Laboratory Investigation, 100:887-899; https: / / doi.org / 10.1038 / s41374-020-0406-7 (c)Masahito Watanabe, Kazuaki Nakano, Hitomi Matsunari, Taisuke Matsuda, Miki Maehara, Takahiro Kanai, Mirina Kobayashi, Yukina Matsumura, Rieko Sakai, Momoko Kuramoto, Gota Hayashida, Yoshinori Asano, Shuko Takayanagi, Yoshikazu Arai, Kazuhiro Umeyama, Masaki Nagaya, Yutaka Hanazono, Hiroshi Nagashima (2013) Generation of Interleukin-2 Receptor Gamma Gene Knockout Pigs from Somatic Cells Genetically Modified by Zinc Finger Nuclease-Encoding mRNA. PLOS ONE, Volume 8, Issue 10, e76478 The gene cassette can be introduced into the safe harbor region and the genetically modified porcine cells can be established according to the methods described in the above-mentioned documents (A) to (C).

[0047] (Genetically modified cloned pigs) In the first aspect, there are no particular limitations on the somatic cell cloning (somatic cell cloning technology) used to obtain a genetically modified cloned pig (gene knock-in cloned pig) from the above-obtained (established) genetically modified pig cells (gene knock-in cells). Somatic cell cloning technology is a technique in which pig cells (preferably genetically modified pig cells) from which a clone is to be produced are cultured to create donor cells, and the nucleus of this donor cell is transferred to a recipient enucleated egg (oocyte) by fusion methods such as cell fusion, and then after cultivation, the donor cell is transferred to a foster parent and fertilized to produce a clone.

[0048] Specifically, in the first nuclear transfer, the above-obtained (established) genetically modified porcine cells (gene knock-in porcine cells) can be introduced into a mature enucleated unfertilized egg. For this introduction, cell fusion using Sendai virus or electrical stimulation or intracytoplasmic injection can be used. Then, an activation treatment can be performed to form a nucleus. Examples of the activation treatment include strontium, ethanol, electrical stimulation, etc. This nucleus can be introduced into an enucleated fertilized egg as a donor for the second nuclear transfer. The above operations may or may not be performed freehand (eg, freehand under a microscope by an experienced technician), but may also be performed using a micromanipulator. The resulting engineered eggs derived from the genetically modified pig cells can be cultured, and when they develop into transplantable embryos, they can be transplanted into the uterus of a foster parent to produce genetically modified cloned pigs (gene knock-in cloned pigs) derived from the genetically modified pig cells (gene knock-in pig cells).

[0049] Furthermore, as described above, the above documents (i) to (iii) describe the production of genetically modified cloned pigs by somatic cloning of genetically modified established pig cells, and the production of genetically modified cloned pigs by somatic cloning of genetically modified established pig cells can be carried out in accordance with the methods described in the above documents (i) to (iii).

[0050] As the above-mentioned recipient enucleated eggs (oocytes), recipient enucleated eggs of any mammal such as pig, cow, horse, sheep, goat, primate (e.g., human, ape (chimpanzee, monkey, etc.)), rodent (e.g., mouse, rat) are preferred, recipient enucleated eggs of mammals whose physical characteristics, etc., are closer to humans than rodents are more preferred, recipient enucleated eggs of pig, sheep, goat, or primate (e.g., human, ape) are even more preferred, and recipient enucleated eggs of pig are particularly preferred. The foster parent into which the above-mentioned transplantable embryo is transplanted is preferably any mammal such as a pig, cow, horse, sheep, goat, primate (e.g., human, ape (chimpanzee, monkey, etc.)), rodent (e.g., mouse, rat), etc., more preferably a mammal whose physical characteristics, etc., are closer to humans than rodents, further preferably pig, sheep, goat, primate (e.g., human, ape), and particularly preferably pig.

[0051] (Sperm from genetically modified cloned pigs) Sperm (sperm of gene knock-in cloned pig) to be used for gene transfer by intracytoplasmic sperm injection, as described below, can be obtained or collected by any method from the genetically modified cloned pig (gene knock-in cloned pig) obtained above.

[0052] (Vector (1)) In the above vector (1) used for gene transfer by intracytoplasmic sperm injection described later in the first embodiment, examples of the specific organ or tissue include internal organs (e.g., pancreas, kidney, ureter, bladder, liver, heart, stomach, intestine, etc.), reproductive organs (e.g., ovaries, testes, etc.), fertilized eggs, embryos, fetuses, bone marrow (e.g., hematopoietic organs), brain, eyes, nose, mouth, skin, nerves, or tissues derived therefrom, or artificial tissues (cell sheets such as cartilage cell sheets, organoids, etc.), with internal organs, reproductive organs, fertilized eggs, embryos, and fetuses being preferred, internal organs and reproductive organs being more preferred, and pancreas (e.g., pancreatic islets), kidney (e.g., metanephros, particularly urinary organs including metanephros, ureters, and bladder) being even more preferred.

[0053] In the above vector (1), the promoter expressed in the specific tissue or organ is not particularly limited as long as it is a promoter expressed in the specific tissue or organ, and examples thereof include a promoter that controls the transcription of Six2 (hereinafter also simply referred to as the "Six2 promoter") or a promoter that controls the transcription of Sall1 (hereinafter also simply referred to as the "Sall1 promoter"), the Six2 promoter, the FGF20 promoter, etc., and the Six2 promoter or the Sall1 promoter is preferred. Both Six2 and Sall1 are known to be expressed, for example, in the kidney, and in particular in renal progenitor cells, particularly in the metanephric mesenchyme region, and more particularly in progenitor cells in the metanephric mesenchyme region. Six2 is known to be expressed in particular in the undifferentiated metanephric mesenchyme region surrounding the tip of the ureteric bud.

[0054] Examples of the vector (1) in the first embodiment include a plasmid vector, a virus vector, and an artificial chromosome vector, and a plasmid vector or an artificial chromosome vector is preferable. The plasmid vector and the viral vector may be any cloning vector. For example, the pCR4Blunt-TOPO vector (Thermo Fisher Scientific) is a commercially available cloning vector that may be used in the present invention. Examples of artificial chromosome vectors include bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), P1 phage-derived artificial chromosomes (PAC), and porcine artificial chromosomes, with BAC, YAC, and PAC being preferred. The above vector (1) may be linear or circular, but is preferably linear from the viewpoint of use in the below-described ICSI and amplification. Linearization can be performed by any method.

[0055] (Process for introducing genes by intracytoplasmic sperm injection) Further gene introduction (second gene modification) can be performed using the vector (1) via intracytoplasmic sperm injection (also called intracytoplasmic sperm injection; ICSI) between an egg (preferably an ovum, more preferably a mature ovum) and a liquid containing the sperm of the above genetically modified cloned pig (sperm of the gene knock-in cloned pig) and the above vector (1) (ICSI mediated gene transfer method). The intracytoplasmic sperm injection can be performed by injecting a liquid containing the sperm and the vector (1) into the egg (preferably a mature egg) by any method (e.g., injection, glass tube, capillary tube) to allow fertilization. The injection procedure may or may not be performed freehand (eg, freehand under a microscope by an experienced technician), but may also be performed using a micromanipulator. The resulting fertilized eggs may or may not be activated by sonication. The fertilized eggs can then be implanted into the uterus of a recipient female (foster mother) to generate the desired transgenic pig (double genetically modified pig) individual.

[0056] The liquid containing the sperm and the vector (1) is not particularly limited as long as it allows the sperm and the vector (1) to coexist, and examples of the liquid include any aqueous solution, any buffer solution, any culture medium, etc., having a pH of about 7.0 to 8.0, which may optionally contain inorganic salts, buffering agents, nutrients, serum albumin, etc. The pig eggs to be subjected to the above-mentioned ICSI are preferably ova, more preferably mature ova.

[0057] <Transgenic pigs> A third aspect of the present invention includes a promoter that is expressed in a specific tissue or organ, and a gene encoding the above-mentioned recombinase that is downstream of and under the control of the promoter that is expressed in the specific tissue or organ, The transgenic pig comprises a gene encoding a functional protein and a gene cassette containing a construct that enables the functional protein to be expressed by the recombinant enzyme. The "promoter expressed in a specific tissue or organ, and the gene encoding the recombinant enzyme present downstream and under the control of the promoter expressed in a specific tissue or organ" may be present (integrated) at any position in the transgenic pig (preferably, cells of the transgenic pig), and may be present (integrated) in, for example, the cytoplasm, genome, mitochondrial genome, etc. of the transgenic pig (preferably, cells of the transgenic pig), and is preferably present (integrated) in the cytoplasm or genome. When present in the cytoplasm, it may be present in the form of a plasmid. In the safe harbor region containing the gene cassette including the construct containing a gene encoding a functional protein, "a promoter expressed in a specific tissue or organ, and a gene encoding the recombinant enzyme that is present downstream of the promoter expressed in a specific tissue or organ and is under its control" may or may not be present (incorporated); however, from the viewpoint of the balance of the expression levels of the control gene cassette and the functional gene cassette, it is preferable that they are not present (not incorporated) in the safe harbor region. The transgenic pig according to the third aspect can be produced by the production method according to the first aspect.

[0058] In the third aspect, specific examples and preferred examples of the "promoter expressed in a specific tissue or organ", "recombinant enzyme", "gene encoding a recombinant enzyme", "functional protein", "gene encoding a functional protein", "construct enabling expression of a functional protein", "gene cassette", and "transgenic pig" include the same specific examples and preferred examples as those described above in relation to the first aspect.

[0059] The transgenic pig (double gene-modified pig) produced by the production method of the first aspect and the transgenic pig of the third aspect may be either a fetus, a young pig, or an adult, but is preferably a fetus from the viewpoint of low immunogenicity.

[0060] As described above, the transgenic pig produced by the production method of the first aspect and the transgenic pig produced by the production method of the third aspect can conditionally express a specific functional protein in a tissue or organ selectively (preferably tissue or organ-specifically) in the tissue or organ. The transgenic pig produced by the production method of the first aspect and the transgenic pig produced by the production method of the third aspect can conditionally kill at least a portion of the cells in the tissue or organ (preferably, the cells inside the tissue or organ excluding the cells on the surface and inner layer of the tissue or organ) when the functional protein is a cytotoxin. This makes it possible to conditionally kill at least a portion of the cells inside only the desired tissue or organ (preferably the cells inside the tissue or organ excluding the cells on the surface and outer layer of the tissue or organ).

[0061] Furthermore, the transgenic pig produced by the production method of the first aspect and the transgenic pig produced by the production method of the third aspect can also express a specific functional protein in a time-specific conditional manner, as described above. The transgenic pig produced by the production method of the first aspect and the transgenic pig produced by the third aspect can also conditionally kill at least a portion of the cells in a tissue or organ (preferably, cells inside the tissue or organ excluding cells on the surface and inner layer of the tissue or organ) in a time-specific manner when the functional protein is a cytotoxin.

[0062] The transgenic pig (double gene modified pig) produced by the production method according to the first embodiment and the transgenic pig according to the third embodiment may or may not be a material for somatic cell cloning. Specifically, somatic cells may be obtained or harvested by any method from the transgenic pig (double gene-modified pig) produced by the production method according to the first embodiment or the transgenic pig according to the third embodiment, and the somatic cells may be used as donor cells. The donor cells may then be nuclear-transferred into a recipient enucleated egg (oocyte) by a fusion method such as cell fusion, and the donor cells may then be cultured and then transplanted and fertilized into a foster parent to produce a clone. The present invention also relates to a transgenic pig (double gene modified pig) produced by the production method of the first aspect, and to somatic cells established (e.g., established or cloned) from the transgenic pig of the third aspect. Since the somatic cells are established, the culture is stable and the cells are highly uniform. As a result, the transgenic pig (double gene-modified pig) produced by the production method according to the first aspect and the clone of the transgenic pig according to the third aspect are excellent in industrial mass production. The transgenic pig (double gene-altered pig) produced by the production method according to the first embodiment and the transgenic pig according to the third embodiment may contain the above clone. By using the transgenic pig (double gene-modified pig) produced by the production method according to the first embodiment or the transgenic pig according to the third embodiment as material for such somatic cell cloning, tissues or organs can be industrially produced from the transgenic pig (double gene-modified pig) produced by the production method according to the first embodiment or the transgenic pig according to the third embodiment.

[0063] <Tissue or organ> A fourth aspect of the present invention relates to a transgenic pig (double gene-modified pig) produced by the production method according to the first aspect, or a tissue or organ extracted by any method from the transgenic pig according to the third aspect. Examples of the organs or tissues include internal organs (e.g., pancreas, kidney, ureter, bladder, liver, heart, stomach, intestines, etc.), reproductive organs (e.g., ovaries, testes, etc.), fertilized eggs, embryos, fetuses, bone marrow (e.g., hematopoietic organs), brain, eyes, nose, mouth, skin, nerves, or tissues derived therefrom, or artificial tissues (cell sheets such as cartilage cell sheets, organoids, etc.), with internal organs, reproductive organs, fertilized eggs, embryos, and fetuses being preferred, internal organs and reproductive organs being more preferred, and pancreas (e.g., pancreatic islets), kidneys (e.g., metanephros, particularly the urinary system including the metanephros, ureters, and bladder) being even more preferred. The tissue or organ according to the fourth aspect is capable of conditionally expressing a specific functional protein within the tissue or organ. When the functional protein of the tissue or organ according to the fourth aspect is a cytotoxin, it is possible to conditionally kill at least a portion of the cells in the tissue or organ (preferably, the cells inside the tissue or organ excluding the cells on the surface and in the outer layer of the tissue or organ).

[0064] In the tissue or organ according to the fourth aspect, when the functional protein is a cytotoxin, at least a portion of the cells (preferably cells inside the tissue or organ excluding cells on the surface and outer layer of the tissue or organ) that have been conditionally killed can be replaced with a foreign substance. Here, the foreign substance means a substance derived from other than the tissue or organ, and includes a substance derived from a human or animal, a drug (e.g., a pharmaceutical product), etc. Examples of the substance include a cell, a growth factor, a hormone, a cytokine, etc., among which a cell is preferable. The cell may or may not be an established cell (e.g., a genetically modified cell, an ES cell, an iPS cell). EXAMPLES

[0065] The present invention will be described in more detail below with reference to examples thereof. However, the present invention is not limited to these examples, and various applications are possible within the scope of the technical concept of the present invention.

[0066] <Reference example 1> (Construction of a gene cassette containing loxP-Venus gene) A knock-in vector was constructed that consists of a splicing acceptor sequence followed by a loxP-STOP-Pause-loxP sequence, downstream of which is Venus-pA and PuroR (puromycin resistance gene cassette) for drug selection. The ROSA region sequences (approximately 700 base pairs each) are linked to both ends (5' and 3') of the vector as homology arms for knock-in. FIG. 1 is a schematic diagram of the gene cassette containing the loxP-Venus gene constructed above. In Figure 1, SA represents a splicing acceptor sequence for allowing the gene cassette to accept transcription from the ROSA promoter, STOP represents a sequence in which three SV40 poly A signal cassettes are linked, Pause represents the human α2 globin gene RNA polymerase II transcriptional pause signal sequence, Venus represents the Venus sequence, pA represents the 3'-UTR (untranslated region) of the rabbit β-globin gene, and PuroR represents a gene cassette composed of the SV40 promoter, puromycin N-acetyltransferase, and pA (poly A) of herpes simplex virus thymidine kinase (HSV-TK).

[0067] <Reference example 2> (Construction of tissue- or organ-specific CreERT2 expression vectors) Approximately 7 kb of bases upstream from the region surrounding the start codon of the mouse Six2 gene were used as the mSix2 promoter, and a gene cassette in which CreERT2 and pA were linked to the 3' end of the mSix2 promoter was introduced into the pCR4Blunt-TOPO vector (Thermo Fisher Scientific) to construct a tissue- or organ-specific CreERT2 expression vector. FIG. 2 is a schematic diagram showing the CreERT2 expression vector constructed above. In FIG. 2, pA indicates the 3'-UTR of the rabbit β-globin gene. The obtained tissue- or organ-specific CreERT2 expression vector was linearized and then used in the ICSI mediated gene transfer method described below.

[0068] <Reference example 3> (Construction of gene cassette containing loxP-DTA gene) A knock-in vector was constructed that consists of a splicing acceptor sequence followed by a loxP-STOP-Pause-loxP sequence, downstream of which is DTA-pA and PuroR (puromycin resistance gene cassette) for drug selection. The ROSA region sequences (approximately 700 base pairs each) are linked to both ends (5' and 3') of the vector as homology arms for knock-in. FIG. 3(a) is a schematic diagram of the gene cassette containing the loxP-DTA gene constructed above. In Figure 3(a), SA represents a splicing acceptor sequence to promote transcription by the ROSA promoter, STOP represents a sequence in which three SV40 poly A signal cassettes are linked, Pause represents a human α2 globin gene RNA polymerase II transcriptional pause signal sequence, DTA represents a diphtheria toxin A fragment sequence, pA represents the 3'-UTR (untranslated region) of the rabbit β-globin gene, and PuroR represents a gene cassette composed of the SV40 promoter, puromycin N-acetyltransferase, and pA (poly A) of herpes simplex virus thymidine kinase (HSV-TK). FIG. 3(b) is a schematic diagram of knock-in of the above gene cassette into the ROSA region.

[0069] (Establishment of cells into which a gene cassette containing the loxP-DTA gene was introduced using CRISPR / Cas9) Pig fetal fibroblasts 5.0×10 5 An RNP complex consisting of a gene cassette containing the loxP-DTA gene, a gRNA complex, and recombinant Cas9 was introduced into the cells by electroporation. Here, the gRNA complex consists of tracrRNA (Trans-activating crRNA) and a porcine ROSA region-gRNA (guide RNA) represented by the following sequence. The following sequence of the porcine ROSA region-gRNA (guide RNA) is located at 65,757,140 to 65,757,159 on the 13th porcine chromosome and is a sequence present between exons 1 and 2 of ROSA. However, the position numbers of the above genes are the position numbers in the UCSC genome browser as of May 24, 2022, and may change due to database updates. Porcine ROSA region-gRNA 5'-GTAGTGCTCTAGATTAGCAC-3' (SEQ ID NO: 8) After selection for puromycin resistance, limiting dilution was performed to less than one cell per well, and genetic analysis was performed on clones derived from the proliferated single-cell cells to establish genetically modified porcine cells in which a gene cassette containing the loxP-DTA gene constructed above was introduced into the porcine ROSA region. FIG. 4 is an electrophoretogram showing the results of confirming the knock-in of the above gene cassette into the ROSA region. As is clear from the results shown in FIG. 4, it was confirmed that the above gene cassette was knocked into the ROSA region.

[0070] (Confirmation of cell death induction by ERT2CreERT2 and tamoxifen in genetically modified porcine cells in which a gene cassette containing the above loxP-DTA gene was introduced into the porcine ROSA region) The ERT2CreERT2 expression vector constructed separately was introduced by electroporation into genetically modified porcine cells in which a gene cassette containing the above-mentioned loxP-DTA gene had been introduced into the porcine ROSA region. The cells were then cultured in a medium containing 10 nM tamoxifen (4-hydroxytamoxifen (4-OHT)). The results are shown in FIG. As is clear from the results shown in FIG. 5, cell death was confirmed in the genetically modified porcine cells into which a gene cassette containing the loxP-DTA gene had been introduced into the porcine ROSA region.

[0071] (Construction of genetically modified cloned pigs from the above genetically modified pig cells) The above-mentioned gene cassette containing the loxP-DTA gene was introduced into the ROSA region of the porcine genetically modified porcine cells, and the cells were cultured in a 35 mm dish at 5 × 10 5 Cells were seeded and subcultured once. Half of the culture medium was removed 14 to 18 hours after subculture, and after 2 hours of culture, the culture medium was collected after gentle pipetting and centrifuged at 1000 rpm for 3 minutes to collect the cells. These were used as nuclear donor cells for nuclear transplantation.

[0072] For nuclear transfer, unfertilized eggs in the metaphase of the second meiosis obtained by in vitro maturation of eggs collected from the ovaries of wild-type female pigs were used as recipient cytoplasm after enucleation. The nuclear transfer procedure was carried out in an egg manipulation solution. Electrical fusion was used to fuse the nuclear donor cells with the recipient cytoplasm. The eggs that had completed the fusion were cultured in a culture solution for 30 minutes to an hour, and then subjected to direct current electrical stimulation. At least some of these eggs were activated normally and began to develop.

[0073] Of the nuclear transfer embryos cultured in vitro, those that cleaved normally on days 1-2, or developed into blastocysts on days 5-6, were transferred into the oviducts or uteruses of recipient pigs whose estrus cycles had been synchronized. At least some of the transplanted embryos gave birth to newborns, and genetically modified cloned pigs were constructed. Sperm were collected from the resulting genetically modified cloned pigs. The sperm can be used in the subsequent ICSI mediated gene transfer method.

[0074] <Reference example 4> (Construction of transgenic pigs using tissue- or organ-specific CreERT2 expression vectors via ICSI) Sperm to be used in ICSI were sonicated to separate the head and tail. The sonication was performed at an intensity that caused separation of the head and tail in approximately 70% of sperm. After co-culturing the tissue- or organ-specific CreERT2 expression vector constructed in the above <Reference Example 2> and pig sperm heads in a culture medium, one sperm head was sucked into an injection pipette, and the injection pipette was injected into an egg using one or more piezo pulses. After confirming that the injection pipette had reached the egg cytoplasm, the sperm head was quickly injected into the egg. Sperm injection was performed in an egg manipulation solution.

[0075] Pre-implantation culture: After ICSI, the sperm-injected eggs were transferred to a PZM5 culture medium drop prepared on a plastic culture dish (IWAKI). After 20 and 44 hours of culture, normal 1-cell stage embryos and normal cleavage embryos were observed. After 116 and 140 hours of culture, blastocyst stage embryos were observed.

[0076] Embryo transfer: Embryos cultured in microdroplets of PZM medium for the above-mentioned period were transferred into the oviduct or uterus of embryo recipient pigs. Normal one-cell embryos and normal cleavage embryos were transferred into the oviduct within one day after ovulation, and blastocysts were transferred into the uterus on the fourth day after ovulation.

[0077] (Sexing test) A total of 42 fetuses were obtained from four recipient pigs (#236, #365, K364, #238). After obtaining tails from the fetuses, genomic DNA was extracted using a DNA extraction kit (DNeasy Blood and Tissue Kit, QIAGEN), and sex determination was performed by PCR using primers targeting amelogenins (AMEL X and AMEL Y). The electrophoretograms of the sex determination results for the 42 animals obtained are shown in FIG. As is clear from Figure 6, #236-1, 5, 6, 8, #365-1, 2, 4, 9, 10, 11, 12, 13, K364-3, 4, 5, 6, 8, and #238-1, 3, 5, 6 were male, while #236-2, 3, 4, 7, 9, #365-3, 5, 6, 7, 8, K364-1, 2, 7, and #238-2, 4, 7 to 12 were male.

[0078] (Gene transfer test) Using the genomic DNA of the 42 fetuses (described above), PCR was performed using a primer targeted to CreERT2 from the mSix2 promoter on the transgene. The PCR products were electrophoresed using 1% agarose gel (Tris-acetate-EDTA) to determine gene transfer. The results are shown in FIG.

[0079] FIG. 7 is an electrophoretic diagram showing the results of gene transfer assessment. In FIG. 7, M is a DNA molecular weight marker, N is a negative control using wild-type pig genomic DNA, and P is a positive control using a CreERT2 expression gene vector. As shown in Figure 7, out of the 42 fetuses, a total of 9 tissue- or organ-specific CreERT2 expression transgenic pig fetuses were obtained, and the production efficiency of transgenic pigs was 21% (#236-2, 5, 6, #365-3, 4, 12, K364-8, #238-5, 7). However, #365-12 and K364-8 were regressed fetuses.

[0080] (Confirmation of CreERT2 expression in fetal metanephroi) Among the fetuses of the above-mentioned tissue- or organ-specific CreERT2 expression gene-transduced pigs, one metanephroi was taken from each of #236-2, 5, 6, #365-3, 4, and 12, and homogenized using a BioMasher (Nippi) in the tissue solution attached to the RNeasy Plus Mini Kit (QIAGEN), and total RNA was collected according to the product protocol. Reverse transcription reaction was performed using SuperScriptIII Reverse Transcriptase (Thermo Fisher Scientific) to obtain cDNA. Amplification reaction was performed using CreERT2 primers and PigSix2 primers using a Veriti thermal cycler (Thermo Fisher Scientific), and CreERT2 expression in the fetal metanephroi was confirmed by RT-PCR (nested PCR). The PCR products were electrophoresed using 1% agarose gel (Tris-acetate-EDTA) to confirm CreERT2 expression in the fetal metanephroi. The results are shown in FIG. CreERT2 primer sequence 1 st : 5'-CATGTCCATGCTGCCCACCTTCG-3' (SEQ ID NO: 9) / 5'-GTATATCCTGGCAGCGATCGC-3' (SEQ ID NO: 10) 2 nd : 5'-TCGCAAGAACCTGATGGACA-3' (SEQ ID NO: 11) / 5'-CGCCGCATAACCAGTGAAAC-3' (SEQ ID NO: 12) PigSix2 primer sequence 1 st : 5'-AGTCGCAGCGTGCTGCGCGAG-3' (SEQ ID NO: 13) / 5'-CTAGGAGCCCAGGTCCACGA-3' (SEQ ID NO: 14) 2 nd : 5'-ACCACGCAGGTCAGCAACTGG-3' (SEQ ID NO: 15) / 5'-TCCGAGCTGCCTAGCACCGAC-3' (SEQ ID NO: 16)

[0081] FIG. 8 is an electrophoretogram showing the results of confirming CreERT2 expression in fetal metanephroi. In FIG. 8, +RT indicates data obtained with reverse transcriptase for sample amplification. As is clear from FIG. 8, CreERT2 gene expression under the mouse Six2 promoter was confirmed in the metanephros of all of the above-mentioned tissue- or organ-specific CreERT2 expression gene-modified pig fetuses (#236-2, 5, 6, #365-3, 4, 12).

[0082] (Determining the copy number of the transgene) Genomic DNA was extracted from the tail tissue of the above-mentioned tissue- or organ-specific CreERT2 expression gene-transduced pig fetuses, #236-2, 5, 6, #365-3, 4, 12, #238-5, and 7, and the copy number of the transgene in the genome was determined by digital PCR. The results are shown in Figure 9. Figure 9 shows the results of transgene copy number determination in the diploid genome, in which β-actin is an internal standard (2 copies).

[0083] As is apparent from the results shown in Figure 9, in the diploid genome, the copy number of the transgene in #236-2 was 5 copies, the copy number of the transgene in #236-5 was 2 copies, the copy number of the transgene in #236-6 was 1 copy, the copy number of the transgene in #365-3 was 16 copies, the copy number of the transgene in #365-4 was 6 copies, the copy number of the transgene in #365-12 was 19 copies, the copy number of the transgene in #238-5 was more than 200 copies, and the copy number of the transgene in #238-7 was 10 copies. As described above, transgenic pig fetuses were obtained in which one or more than two copies, or even more than 200 copies of tissue- or organ-specific CreERT2 gene were introduced per individual (diploid).

[0084] <Example 1> (Establishment of cells into which a gene cassette containing the loxP-Venus gene was introduced using CRISPR / Cas9 in the ROSA region) The RNP complex consisting of the gene cassette containing the loxP-Venus gene, the gRNA complex, and the recombinant Cas9 constructed in <Reference Example 1> was transfected at 5.0 × 10 5 The gene was introduced into porcine fetal fibroblast cells. Here, the gRNA complex consists of tracrRNA (Trans-activating crRNA) and the porcine ROSA region-gRNA (guide RNA) represented by the sequence of SEQ ID NO: 8. The following sequence of the porcine ROSA region-gRNA (guide RNA) is located at 65,757,140 to 65,757,159 on the porcine chromosome 13, and is a sequence present between exons 1 and 2 of ROSA. However, the position numbers of the above genes are the position numbers in the UCSC genome browser as of May 24, 2022, and may change due to database updates. After selection for puromycin resistance, limiting dilution was performed to less than one cell per well, and genetic analysis was performed on clones derived from the proliferated single-cell cells. Genetically modified porcine cells were established in which the gene cassette containing the loxP-Venus gene constructed above was introduced into the porcine ROSA region.

[0085] (Construction of genetically modified cloned pigs from the above genetically modified pig cells) The gene-modified porcine cells obtained by introducing the gene cassette containing the loxP-Venus gene into the porcine ROSA region were plated in a 35 mm dish at 5 × 10 5 Seed the cells and subculture once. 14-18 hours after subculture, remove half of the culture medium. After 2 hours of culture, gently pipette the cells and collect the medium. Centrifuge at 1000 rpm for 3 minutes to collect the cells. These cells will be used as donor nuclei for nuclear transfer.

[0086] For nuclear transfer, unfertilized eggs in the metaphase of the second meiosis obtained by in vitro maturation of eggs collected from the ovaries of wild-type female pigs were used as recipient cytoplasm after enucleation. The nuclear transfer procedure was carried out in an egg manipulation solution. Electrical fusion was used to fuse the nuclear donor cells with the recipient cytoplasm. The eggs that had completed the fusion were cultured in a culture solution for 30 minutes to an hour, and then subjected to direct current electrical stimulation. At least some of these eggs were activated normally and began to develop.

[0087] Of the nuclear transfer embryos cultured in vitro, those that cleaved normally on days 1-2, or developed into blastocysts on days 5-6, were transferred into the oviducts or uteruses of recipient pigs whose estrus cycles had been synchronized. At least some of the transplanted embryos gave birth to newborns, and genetically modified cloned pigs were constructed. Sperm were collected from the resulting genetically modified cloned pigs. The sperm can be used in the subsequent ICSI mediated gene transfer method.

[0088] (Construction of transgenic pigs using tissue- or organ-specific CreERT2 expression vectors via ICSI) The tissue- or organ-specific CreERT2 expression vector constructed in the above <Reference Example 2> and the heads of the pig sperm are co-cultured in a culture medium, and normal development of transgenic pig fetuses is obtained from the embryo recipient pig by the same ICSI procedure, egg culture, and embryo transfer into the uterus of the embryo recipient pig as in the above <Reference Example 4>. By administering tamoxifen to a female pig carrying the above-mentioned normally developed fetus, or by transplanting the metanephroi extracted from the above-mentioned normally developed fetus into the body of an immunodeficient mouse and then administering tamoxifen to the mouse, or by culturing the extracted metanephroi in vitro and adding tamoxifen to the culture medium, Venus fluorescence can be conditionally observed in specific tissues or organs.

[0089] <Example 2> (Construction of transgenic pigs using tissue- or organ-specific CreERT2 expression vectors via ICSI) The sperm of a genetically modified cloned pig into which the tissue- or organ-specific CreERT2 expression vector constructed in <Reference Example 2> above and the gene cassette containing the loxP-DTA gene obtained in <Reference Example 3> above have been introduced are co-cultured in a culture medium, and normally developed fetuses of the transgenic pig are obtained from the embryo recipient pig by the same ICSI procedure, egg culture, and embryo transfer into the uterus of the embryo recipient pig as in <Reference Example 4> above.

[0090] <Comparative Example> (Construction of all-in-one vector 1 containing both the mSix2 promoter and the CreERT2 and loxP-DTA genes) An all-in-one knock-in vector was constructed in which the mSix2 promoter was located approximately 7 kb upstream from the region surrounding the start codon of the mouse Six2 gene, CreERT2 and pA were linked to the 3' end of the mSix2 promoter, a CAG promoter was linked downstream of the pA, a loxP-STOP-loxP sequence was linked to the 3' end of the CAG promoter, and DTA-pA was linked downstream of that. FIG. 10 is a schematic diagram showing the all-in-one vector 1 containing both the mSix2 promoter and the CreERT2 and loxP-DTA genes constructed above.

[0091] (Construction of all-in-one vector 2 containing both the mSix2 promoter and the CreERT2 and loxP-DTA genes) An all-in-one knock-in vector 2 was constructed, which consists of approximately 7 kb upstream of the region surrounding the start codon of the mouse Six2 gene as the mSix2 promoter, P2A (self-cleaving 2A peptide derived from porcine Teschovirus-1) linked to the 3' end of the mSix2 promoter, ERT2CreERT2 and pA linked to it, a CAG promoter linked downstream of the pA, and a DTA-pA sequence (reverse) sandwiched between FLEx switch-type loxPlox2272 and loxPlox2272 (reverse) linked to the 3' end of the CAG promoter. FIG. 11 is a schematic diagram showing the all-in-one vector 2 containing the mSix2 promoter and both the CreERT2 and loxP-DTA genes constructed above. The obtained all-in-one vectors 1 and 2 containing both the mSix2 promoter and the CreERT2 and loxP-DTA genes were linearized and then used in ICSI described below.

[0092] (Construction of transgenic pigs using all-in-one vectors 1 and 2 via ICSI) A transgenic pig was constructed via ICSI in the same manner as in Example 1, except that the "tissue- or organ-specific CreERT2 expression vector" was changed to the all-in-one vector 1 or 2 containing both the mSix2 promoter and the CreERT2 and loxP-DTA genes. The results of constructing transgenic pigs using all-in-one vector 1 or 2 via ICSI are summarized in Tables 1 and 2 below.

[0093] [Table 1] As is clear from Table 1, in transfection using all-in-one vector 1 via ICSI, one normally developed transgenic fetus was obtained, but the production efficiency of transgenic pig fetuses was extremely low, less than 0.35%, which is considered to be due to the imbalance (excessive) of the copy number of the loxP-DTA gene relative to the copy number of the CreERT2 gene.

[0094] [Table 2] As is clear from Table 2, gene transfer using all-in-one vector 2 via ICSI did not result in normal development of gene-transfected fetuses (production efficiency of gene-transfected pig fetuses: 0%). In addition, all gene-transfected fetuses regressed.

[0095] As described above, it was difficult to construct transgenic pigs using an all-in-one vector containing both the mSix2 promoter and the CreERT2 and loxP-DTA genes.

Claims

1. Sperm of a genetically modified cloned pig obtained by somatic cloning of a genetically modified pig cell obtained by introducing a gene cassette containing a gene encoding a functional protein and a construct capable of expressing the functional protein by a recombinase into a safe harbor region; A method for producing a transgenic pig, comprising the step of introducing a gene into an egg by insemination with a liquid containing the vector (1), The vector (1), A gene encoding the recombinase is contained in the downstream of a promoter that is expressed in a specific tissue or organ and is under the control of the promoter that is expressed in the specific tissue or organ, A production method in which the ratio of the copy number of the gene encoding the functional protein to the copy number of the gene encoding the recombinant enzyme is 1 / 2 to 1 / 300.

2. Sperm of a genetically modified cloned pig obtained by somatic cloning of a genetically modified pig cell obtained by introducing a gene cassette containing a gene encoding a functional protein and a construct capable of expressing the functional protein by a recombinase into a safe harbor region; A method for producing a transgenic pig, comprising the step of introducing a gene into an egg by insemination with a liquid containing the vector (1), The vector (1), A gene encoding the recombinase is contained in the downstream of a promoter that is expressed in a specific tissue or organ and is under the control of the promoter that is expressed in the specific tissue or organ, The method for producing said functional protein is at least one selected from the group consisting of metabolic enzymes, cell proliferation-related factors, growth-related gene proteins, cytotoxins, and suicide gene proteins.

3. A gene cassette including a construct that includes a gene encoding a functional protein and enables expression of the functional protein by a recombinase; The method according to claim 1 or 2, which has improved production efficiency compared to a method for producing a transgenic pig, which includes a gene transfer step of microinsemination into an egg a vector containing both a promoter expressed in a specific tissue or organ and a gene cassette containing a gene encoding the recombinant enzyme that is located downstream and under the control of the promoter expressed in a specific tissue or organ.

4. The method according to claim 1 or 2, wherein the promoter is a Six2 promoter, a Sall1 promoter or an FGF20 promoter.

5. The method according to claim 1 or 2, wherein the introduction of the gene cassette into the safe harbor region is by genome editing.

6. The method of claim 1 or 2, wherein the safe harbor region is a ROSA region.

7. The method according to claim 1 or 2, wherein the vector (1) is a plasmid vector or an artificial chromosome vector.

8. A somatic cell cloning source comprising a genetically modified porcine cell comprising a gene encoding a functional protein, the gene cassette comprising a construct capable of expressing the functional protein by a recombinase in a safe harbor region, A somatic cell cloning material for providing a material for gene transfer by intracytoplasmic sperm injection in the presence of the following vector (1). (1) A vector comprising a promoter expressed in a specific tissue or organ, and a gene encoding the recombinase that is present downstream of and under the control of the promoter expressed in a specific tissue or organ, wherein the ratio of the copy number of the gene encoding the functional protein to the copy number of the gene encoding the recombinase is 1 / 2 to 1 / 300.

9. A somatic cell cloning source comprising a genetically modified porcine cell comprising a gene encoding a functional protein, the gene cassette comprising a construct capable of expressing the functional protein by a recombinase in a safe harbor region, A somatic cell cloning material for providing a material for gene transfer by intracytoplasmic sperm injection in the presence of the following vector (1): A somatic cell cloning source material, wherein the functional protein is at least one selected from the group consisting of metabolic enzymes, cell proliferation-related factors, growth-related gene proteins, cytotoxins, and suicide gene proteins. (1) A vector comprising a promoter that is expressed in a specific tissue or organ, and a gene encoding the recombinase that is downstream of and under the control of the promoter that is expressed in a specific tissue or organ.

10. A gene cassette including a construct that includes a gene encoding a functional protein and enables expression of the functional protein by a recombinase; The somatic cell cloning material described in claim 8 or 9, which has improved production efficiency by somatic cell cloning compared to a somatic cell cloning material containing genetically modified pig cells containing both a promoter expressed in a specific tissue or organ and a gene cassette containing a gene encoding the recombinant enzyme that is located downstream of the promoter expressed in a specific tissue or organ and under its control.

11. 10. The somatic cell cloning material according to claim 8 or 9, wherein the promoter is a Six2 promoter, a Sall1 promoter or an FGF20 promoter.

12. A gene encoding the recombinase is contained in the downstream of a promoter that is expressed in a specific tissue or organ and is under the control of the promoter that is expressed in the specific tissue or organ, A transgenic pig comprising a gene encoding a functional protein, a gene cassette comprising a construct that enables the functional protein to be expressed by the recombinant enzyme, in a safe harbor region, and a ratio of the copy number of the gene encoding the functional protein to the copy number of the gene encoding the recombinant enzyme of 1 / 2 to 1 / 300.

13. A gene encoding the recombinase is contained in the downstream of a promoter that is expressed in a specific tissue or organ and is under the control of the promoter that is expressed in the specific tissue or organ, A gene cassette including a construct that includes a gene encoding a functional protein and enables expression of the functional protein by the recombinase is included in the safe harbor region; The transgenic pig, wherein the functional protein is at least one selected from the group consisting of a metabolic enzyme, a cell proliferation-related factor, a growth-related gene protein, a cytotoxin, and a suicide gene protein.

14. A gene cassette including a construct that includes a gene encoding a functional protein and enables expression of the functional protein by a recombinase; The transgenic pig according to claim 12 or 13, which has an improved survival rate compared to a transgenic pig produced by a production method including a gene transfer step of injecting an egg with a vector containing both a promoter expressed in a specific tissue or organ and a gene cassette containing a gene encoding the recombinant enzyme that is located downstream and under the control of the promoter expressed in a specific tissue or organ.

15. The transgenic pig according to claim 12 or 13, wherein the promoter is a Six2 promoter, a Sall1 promoter or an FGF20 promoter.

16. A somatic cell established from the transgenic pig according to claim 12 or 13.

17. A tissue or organ isolated from the transgenic pig according to claim 12 or 13.

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