The Hidden!: Humanized mouse

4 Sept 2018

Humanized mouse

Humanized mouse

https://patents.google.com/patent/WO2013145331A1/en

Humanized mouse

Abstract

Provided are: an embryonic stem cell produced by culturing an embryo from an immunodeficient mouse, which lacks both Rag2 gene and Jak3 gene, in the presence of a GSK3 inhibitor and an MEK inhibitor; and a transgenic mouse produced using the embryonic stem cell.

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A01K67/0278 Humanized animals, e.g. knockin

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WO2013145331A1

WO Application

Find Prior Art SimilarOther languagesFrench Japanese Inventor山村研一荒木喜美岡田誠治下野明彦 Original Assignee株式会社トランスジェニック国立大学法人熊本大学 Priority date 2012-03-27

Family: US (1)EP (1)JP (1)KR (1)CN (1)CA (1)WO (1)

DateApp/Pub NumberStatus

2012-03-27PCT/JP2012/058790

2013-10-03WO2013145331A1ApplicationInfoPatent citations (9) Non-patent citations (23) Cited by (2) Legal events Similar documents Priority and Related Applications External linksEspacenet Global Dossier PatentScope Discuss

Description

Humanized mouse

The present invention relates to a mouse embryonic stem cells harvested from immunodeficient mice (ES cells) and liver humanized.

Liver metabolism, discharge, detoxification, maintenance of homeostasis of body fluid is organ plays a central role in the body in such. The liver is an organ that only play in vivo, even when cut about 80% of the total weight, are known to have the ability to play up to the original weight.

Since the function of the liver variety, although the liver many genes are expressed, that amount, genetic disease there are many due to abnormal gene expression in the liver.

If abnormal liver function by liver disease or the like occurs, there may be no effective treatment other than liver transplantation. Therefore, the need for prediction of human blood metabolite was higher in developing early stage. To predict blood metabolites and hepatic function properly is liver is a need to develop humanized animals.

Conventional relates Preparation of model mice of human liver, for example, Heckel et al., Transgenic albumin (Alb) plasminogen activator urokinase-type promoter (Plau) construct linked gene (Alb-Plau) was introduced (reported on Tg (Alb-Plau) (non-Patent Document 1: Heckel et al.Cell 62: 447-456,1990)) mice, however, the mice die in bleeding intestine such as day 4 after birth It can not be used in the experiment for. In contrast, in the Tg (Alb-Plau), survive successfully established individual lineages, hepatocytes is reported an example in which the liver is regenerated by hepatocytes lacking Alb-Plau gene into dividing They are (non-patent document 2: Sandgren et al.Cell 66: 245-256,1991). Also, mice construct linked to the lacZ gene in the metallothionein promoter was introduced, namely, adult hepatocytes of transgenic mice were labeled liver cells of donor lacZ is a marker gene (Tg (MT-nLacZ) mice) Tg were transplanted into (Alb-Plau), successful example is (non-Patent Document 3: Rhim et al.Science 263: 1149-1152,1994).

Furthermore, there is an example of performing the transplantation of human hepatocytes into immunodeficient mice. For example, Rag2 - / - respect to gene-deficient immunodeficient mice transplanted with liver cells, an example of performing infection experiments hepatitis B virus (HBV) (Non-Patent Document 4: Dandri et al.Hepatology 33: 981-988, 2001), or Tg transplanted with human hepatocytes (crossed the SCID is Alb-Plau) and immunodeficient mice, SCID mice became immunodeficiency (Tg (Alb-Plau)) (Tg (Alb-Plau) ; SCID)), examples of performing infection experiments of hepatitis C virus (non-Patent Document 5: Mercer et al.Nature Med.7: 927-933,2001), and the like.

Furthermore, Tateno et al., Albumin enhancer / promoter urokinase plasminogen activator transgenic mice with impaired liver (uPA mice) multiplied by a SCID mouse, uPA / SCID transgenic is either transformed even homozygotes to prepare a mouse (non-Patent Document 6: Tateno et al.Amer.J.Pathol 165: 901-912,2004). In this report, human hepatocytes Tg; have been described for the implantation process improvements in the (Alb-Plau SCID), mortality in high chimera by eliminating the influence of complement from human liver cells by Futhan treatment It is lowered.

Furthermore, studies have demonstrated the potential for gene therapy of immunodeficient mice lacking Rag2 gene as a model has also been reported (Non-Patent Document 7: Orthopedic disorders Series IV somatic viewed from shaping and disaster Surgery "molecular cloning technology and regenerative medicine, "Vol.45, NO.11, PAGE.1040-1041,2002).

However, In these mouse models, the remaining mouse liver cells, a host, 100% of the cells is not a hepatocyte model substituted with cells derived from human. Moreover, not necessarily human-derived cells is reproduced, forced transplanted cells derived from human. Moreover, the mouse-derived liver cells are still present, insufficient verification of human liver function.

On the other hand, for the establishment of NOG mouse-derived ES cell lines for transmission to the germ line, by using the differentiation signal inhibitor (PD0325901, CHIR99021), an attempt to establish the ES cells have also been made (Non-Patent Document 8: Japanese Experiment Zoological Society meeting Abstracts Vol.58th, Page 210,2011).

However, NOG mice in terms difficult breeding, it is difficult to obtain a large number of mice for experiment.

Heckel et al. Cell 62: 447-456,1990 Sandgren et al. Cell 66: 245-256,1991 Rhim et al. Science 263: 1149-1152,1994 Dandri et al. Hepatology 33: 981-988,2001 Mercer et al. Nature Med. 7: 927-933,2001 Tateno et al. Amer. J. Pathol 165: 901-912,2004"orthopedic diseases Series IV somatic cell clone technology and regenerative medicine as seen from the molecular level," Vol shaping and disaster surgery. 45, NO. 11, PAGE. 1040-1041,2002 Japan Association for Laboratory Animal Science Meeting Abstracts Vol. 58th, Page 210,2011

The present invention is immunodeficient mouse embryonic stem cells taken from (ES cells) and liver and to provide a humanized mouse.

The present inventor has conducted extensive studies to solve the above problems, Rag2 gene and Jak3 gene from both deficient immunodeficient mouse embryos, which can be produced an optimal mouse human hepatocytes transplanted embryos It found that sex stem cells are obtained, and have completed the present invention.

That is, the present invention is as follows.

(1) Rag2 gene and immunodeficient mouse embryos Jak3 gene together deficient embryonic stem cells obtained by culturing in the presence of a GSK3 inhibitor and a MEK inhibitor.

(2) receipt number is given by NITE ABP-1297, embryonic stem cell according to (1).

(3) estrogen receptor gene and diphtheria toxin gene is introduced, embryonic stem cell according to (1) or (2).

(4) growth hormone gene inherent in the cell is replaced with one derived from human embryonic stem cells described in (3).

(5) In addition, drug-metabolizing enzyme gene inherent in the cell is replaced with one derived from human embryonic stem cell according to (4).

(6) drug metabolizing enzyme gene inherent in cells, Cyp3a11, Cyp3a13, Cyp3a25, and embryonic stem cell according to at least one selected from the group consisting of Cyp3a41 (5).

(7) (1) or mice generated using embryonic stem cell according to (2).

(8) the (3) to have been generated using embryonic stem cell according to any one of (6), the transgenic mice.

(9) cause liver cell damage by administration of an antiestrogen, mouse described in (8).

(10) wherein with transplanting human hepatocytes into mice as described in (7), a mouse, characterized in that the removal of the said mouse-derived hepatocytes by administering an antiestrogen, the liver has been humanized.

(11) mice according to the human-derived hepatocytes are derived from patients with liver disease (9).

(12) a mouse according to (10), human liver disease model mice.

(13) Rag2 gene and Jak3 gene both deficient immunodeficient mouse embryos, characterized by culturing in the presence of a GSK3 inhibitor and a MEK inhibitor, a manufacturing method of embryonic stem cells from immunodeficient mice.

(14) characterized in that said administering an antiestrogen in mice according to (8), the method producing liver injury model mice.

(15) wherein with transplanting human hepatocytes into mice as described in (8), and removing the murine hepatocytes by administering an antiestrogen, the production of mouse liver humanized Method.

(16) human hepatocytes are derived from patients with liver disease, the method described in (15).

The present invention, embryonic stem cells for the establishment of optimal murine to human liver cell transplantation is provided. Embryonic stem cells of the invention can be introduced various human gene for liver function, it is possible to establish a humanized liver model mice. Therefore, mice were established from embryonic stem cells of the present invention can be utilized in human liver cell transplantation, it is very useful in that it allows 100% of the humanized.

Figure 1 is a diagram showing a variant lox.

Figure 2 is a diagram showing a Dre / rox system.

Figure 3 is a construction diagram of a replacement vector for introducing a human growth hormone gene in ES cells.

Figure 4 is a construction diagram of a replacement vector for introducing a human drug-metabolizing enzyme gene in ES cells.

Figure 5 is a diagram illustrating the course of the introduction into ES cells diphtheria toxin gene to mouse hepatocytes death.

Figure 6 is a diagram showing a portion of transplanting human hepatocytes into mouse embryos.

Figure 7 is a diagram showing the embryos of mice transplanted with human hepatocytes.

S1: shows the intraperitoneal administration of anesthetics, S2: figure incised abdominal fetal exposed outside peritoneal A: cell yolk 嚢静 veins to be injected, B: cell injection site (yellow arrows), C : (looks blue) liver after injection of the blue dye, D: excised liver (left liver after the blue dye injection, the right is not injected with dye liver)

Figure 8 is a diagram showing the hepatocytes induced to differentiate from iPS cells.

Figure 9 is a diagram showing the hepatocytes induced to differentiate from iPS cells.

The present invention will be described in detail.

1. SUMMARY The present invention is an embryonic stem cell established from both deficient immunodeficient mice Rag2 gene and Jak3 gene from the embryonic stem cells, is that the liver has established humanized mice.

In the present invention, the embryos collected from immunodeficient mice by culturing in the presence of a GSK3 inhibitor and a MEK inhibitor, has succeeded in establishing ES cells.

Rag2 and deficient BALB / c mice Jak3 (BALB / c; Rag2 - / -; Jak3 - / -: called "BRJ mouse") is deficient T cells, B cells, NK cells and NKT cells, BALB / c are immunodeficient mice with genetic background. When transplanting human cells in the mice, therefore the cells to engraft in mice body mice produced will that humanized mice at the cellular level.

However, such humanized mice, since the mouse-derived cell is a host remains, not organs replaced all of human origin, are not necessarily optimized in performing functional analysis and research of the organ was a mouse that it is not necessarily. Further, in making the optimization mouse, it is necessary to perform the various genetic modification can not be performed using individual mouse.

Therefore, in the present invention, 100% of the cells to establish the mice with humanized liver, to establish the optimal genetically modified mice to humanization. The genetically modified mice As a result of intensive studies to aim humanized from early ontogeny, and succeeded in establishing embryonic stem cells from BRJ mice (hereinafter referred to as "ES cells"). As ES cells was used to generate chimeric mice, it was also successfully produced germ chimeric mice transmitted to germline. Next, to confirm the safety with to maintain the liver function over a long period of time, to establish the mice with normal human liver. Furthermore, in order to analyze the condition as well as established disease models same symptoms as human patients with liver disease, to establish a mouse having a human mutation liver. Further, establishing an optimized model mice to human disease to develop versatile novel therapies.

2. Immunodeficient mice made use of BRJ mice are mice Rag2 gene and Jak3 gene is both knockout is a mouse that has been previously established (Ono A, Hattori S, Kariya R, Iwanaga S, Taura M, Harada H, Suzu S, Okada S.Comparative study of human hematopoietic cell engraftment into BALB / c and C57BL / 6 strain of rag-2 / jak3 double-deficient mice.J Biomed Biotechnol 2011: 539748,2011).

This mouse can be obtained by mating with a Rag2 deficient mice and Jak3 deficient mice.

Rag (Recombination activating gene) 2 genes have an essential function in the reconstruction of the immunoglobulin genes and T-cell receptor expressed in immature lymphocytes, is the essential gene in the maturation of T cells and B cells .

Preparation Method of mice this Rag2 gene has been knocked out, and more information about the gene, Shinkai Y. et al. , Cell. 1992 Mar 6; 68 (5): 855-67, Chen J. et al. , Curr Opin Immunol. 1994 Apr; 6 (2): 313-9 has been described, for example, general methods well known in the art to, for example, by a method of using a targeting vector can be produced (Capecchi, M.R.,. Science, (1989) 244,1288-1292). This method is using homologous recombination with the gene on Rag2 gene targeting vector in mouse ES cells.

Jak3 (Janus kinase 3) is a non-receptor tyrosine kinase, IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21 in a cell of the common γ chain that is common to receptors the signals through the common γ chain associate the region is a protein having a function of transferring into the cell. Common γ chain, since the signal through the Jak3 is an essential signal to NK cells, NK cells this pathway is injury will be deficient. In other words, when knocking out Jak3 gene, it can abolish the NK activity.

Jak3 gene and details of the common γ chain genes, Park SY. et al. , Immunity. 1995 Dec; 3 (6): 771-82, Suzuki K. et al. , Int Immunol. 2000 Feb; 12 (2): such as are described in 123-32, with reference to these documents, Jak3 gene deficient, it is possible to obtain a mouse NK activity was lost.

It should be noted, Rag2 deficient (- / -) mice and Jak3 deficient (- / -) mice, can also be obtained from Kumamoto University Life Resources Research and Support Center. And these mice by backcrossing a commercial BALB / c mice, BALB / c Rag2 deficient with genetic background of Balb / c mice of the same strain (- / -) mice and BALB / c Jak3 deficient ( - / -) mice can be obtained respectively.

Rag2 gene double knockout mice that both and Jak3 gene deficient prepared, first, to obtain a F1 by mating the BALB / c Rag2 deficient mice and BALB / c Jak3 deficient mice, then crossed F1 together with F2 get the mouse. Then, Rag2 deficient from the (- / -) and Jak3 deficient (- / -) of may be selected both deficient mice (BRJ mice). As a selection method of BRJ mice that are deficient in both genes, for example, Rag2 and Jak3 can be confirmed by PCR or Southern blotting.

3. ES cells established the present invention ES cells, by culturing the embryos collected from BRJ mice obtained as described above in the presence of a GSK3 inhibitor and a MEK inhibitor, it can be obtained.

First, whether obtained by culturing fertilized or 2-cell embryos from BRJ female mice after fertilization, or obtain blastocysts directly. Fertilization natural mating, or by in vitro fertilization method. In vitro fertilization is performed by culturing the oocytes obtained superovulated female mice, and sperm collected from male mice.

Then, the collected blastocyst method or inner cell mass in a medium for animal cell culture, about 1-3 weeks in the presence of GSK-3 inhibitors and MEK inhibitor, preferably cultured 14-18 days.

GSK-3 (glycogen synthase kinase 3) is a serine / threonine protein kinase, an enzyme acting production and apoptosis of glycogen, a number of signaling pathways involved in such maintenance of stem cells. The GSK-3 inhibitors, CHIR99021 (get: Wako Pure Chemical), 6-Bromoindirubin-3-oxime (BIO) (get: Wako Pure Chemical) and the like. Amount into the medium of GSK-3 inhibitors, 0.1 ~ 10 [mu] M (micromolar), preferably 0.3 ~ 3 [mu] M. It is not particularly limited in time to addition to the medium of GSK-3 inhibitors, but it is preferably added from the start of the blastocyst method culture.

MEK inhibitors inhibit MAP Kinase Kinase (MEK) activity, a suppressing protein kinase inhibitor activation ERK1 / ERK2. The MEK inhibitors such PD0325901 (get: Wako Pure Chemical), U0126 (get: Promega), and the like. The addition amount of the medium in PD0325901 inhibitor is not intended to be limited, for example, 3 [mu] M.

Culture conditions are not limited, for example, about 37 ° C., carried out in a 5% CO 2 atmosphere. Subculture in 3-4 day intervals, or may be carried out on plates coated with mouse embryonic fibroblast (MEF) cells feeder or collagenase I.

As the medium, for example GMEM medium (Glasgow's Minimal Essential Medium), DMEM (Dulbecco's Modified Eagle's Medium), and the like RPMI1640 medium medium. The culture medium, KSR (Knockout Serum Replacement), fetal bovine serum (FBS), basic fibroblast growth factor (bFGF), beta-mercaptoethanol, nonessential amino acids, glutamate, sodium pyruvate and antibiotics (e.g., penicillin , streptomycin, etc.) selected from the like can be appropriately added.

After a predetermined period the culture, and recovering the ES cells by incubation in media containing EDTA or collagenase IV. Recovered ES cells can also be multiple passages by culturing in the presence or absence of feeder cells, if necessary. Incidentally, the culture of inner cell mass in a feeder-free conditions can be carried out in medium conditioned by MEF.

ES cells cultured may be identified generally using these marker genes. The marker gene of ES cells, for example, Oct3 / 4, alkaline phosphatase, Sox2, Nanog, GDF3, REX1, FGF4, and the like. The presence of the marker gene or gene product may be detected by any technique such as PCR or Western blotting.

Further, whether or not ES cells of the present invention is of interest is the detection whether the BALB / c of SNP markers, whether a Rag2 deficient and Jak3 deficiency, analysis by PCR or Southern blotting It can also be confirmed by. For example, the mouse of the SNP database is http: // www. broadinstitute. org / snp / have been published in the mouse, can see that by using this database is BALB / c if collating SNP information, if deficient Rag2 and Jak3 gene is the ES cells of the present invention it is determined that the.

ES cells obtained in this way is referred to as "BRJ8", with the date 2012, March 23 (date of receipt), National Institute of Technology and Evaluation Patent Microorganisms Depositary Center (Yubinbango292-0818 Kisarazu City, Chiba Prefecture It was an international deposit based on the Budapest Treaty on Kazusa legs bowed in 2-5-8 NITE Department of biotechnology Patent microorganisms Depositary Center). The receipt number is a "NITE ABP-1297".

More information of the ES cells are as follows.

Have been previously published papers for establishment method of ES cells by GSK inhibitor and a MEK inhibitor, ES cells obtained are inherited genetic properties of the original strain, respectively include features specific there. Immunodeficient mice there are various systems, but in addition to the original genetic background, since they have individually mutations, has a unique characteristic for each lineage. For example, NOG mice with reported, the NOD system, and has a defect in common gamma (IL2R-γ) genes SCID and IL2 receptor. NOD mice have originally deficient in the complement. Gene responsible for SCID are Prkdc (DNA-dependent protein kinase, catalytic subunit), this gene is a gene necessary for reconstruction of the immunoglobulin genes and T cell receptor. Therefore, if there is a mutation in this gene, it becomes impossible to B lymphocytes and T lymphocytes. Further, the IL2R-gamma is a common molecules constituting the IL-2, IL-4, IL-7, IL-9, interleukin receptor such as IL-15 and IL-21. Therefore, this molecule is lost, the signal is no longer transmitted through these interleukins can not immune responses. Taken together, complement, B lymphocytes, in addition to defects in T lymphocytes old, hypofunction of macrophages and dendritic cells also seen, immunocompromised becomes more severe. However, or worked fatal in some cases because it is a severe immunodeficiency even opportunistic infection pathogens that are not at all a problem in normal mice, thymoma occurs in a high rate.

Meanwhile, BRJ mice, which genes of Rag2 and Jak3 lineage of BALB / c mice deficient, graft survival of the transplanted cells are known in good. Rag2 the genes necessary for reconstruction of similarly immunoglobulin genes or T cell receptor and Prkdc, also, Jak3 is a gene present downstream of IL2R-gamma, thus, immunodeficiency severe by a deficiency of these genes the state. However, BRJ is relatively easy breeding and propagation.

In preliminary experiments, tried to establish ES cells with GMEM-KSR medium is conventionally used, it can only be established very bad strain growth, approximately 50% to produce chimeric mice with this strain only obtained of chimerism, it did not contribute to the germline. In contrast, in the present invention, the addition of GSK3 inhibitor and a MEK inhibitor that is effective for the undifferentiated state maintenance of ES in the medium, it was possible to establish ES cells of interest. ES cells of the present invention has high viability, chimerism is high. The reason is, as compared to ES cells generated using conventional methods, because is maintained well undifferentiated state. One of the important signal that acts on the differentiation of ES cells is the path of the ERK / MEK which via a FGF receptor from FGF4. That, ERK serves as a signal differentiation. Furthermore, GSK-3 stimulates Wnt signaling by phosphorylating β- catenin, induce differentiation. Thus, by using two inhibitors PD0325901 and GSK3 inhibitors that are potent MEK inhibitors (2i), ES cells of the present invention can maintain pluripotency suppress differentiation.

4. To establish the optimal genetically modified mice to genetic modification humanization rather than the mouse became adult, after the endogenous gene in the ES cell stage were introduced either replace the human, or a human gene in ES cells, it is necessary to produce a mouse from the genetic modification and / or transgenic ES cells.

Therefore, in the present invention, by introducing a gene of interest in ES cells, or to replace a gene endogenous to ES cells of the human gene, Cre-loxP system is a recombinant system from bacteriophage, recombinant derived Vibrio system in which VCre-Vlox system utilizes Dre / rox system or homologous recombination by the system obtained by modifying these recombination systems, a recombinant system utilizing homologues of Cre.

1oxP (locus of crossing (X-ring) over, P1) is 'a sequence consisting of (SEQ ID NO: 1), 5 34 bases (5'-ATAACTTCGTATAGCATACAT TATACGAAGTTAT-3)' that ends 13 bases (inverted repeat sequence 1 ), and 3 'end from 13 base sequence of (as inverted repeat sequence 2), constitutes the inverted repeat sequences, respectively, in sequence 8 called base spacer the inverted repeat sequence 1 and 2, indicated by "GCATACAT" It is sandwiched (Figure 1). The "inverted repeat sequence", the sequence of one side of the spacer as a boundary is, and the sequence of the other side, means a sequence that is complementary to a direction facing each other.

The Cre (Causes recombination) means a recombinant enzyme that causes genetic recombination (also referred to as recombinase) recognizes the repetitive sequences, which cleaves a "cataca" spacer unit in cutting fashion to sticky ends.

Incidentally, in the bacteria, it occurs recombination between the two locations 1OxP, insertion or deletion reaction occurs (Fig. 1). If it is possible to cause the insertion reaction in mammalian cells, it is possible to insert any gene after, applicability is dramatically widened. Is large nucleus in mammalian cells, once circular DNA having deleted loxP is will diffuse, insertion reaction is hardly observed.

Accordingly, the present inventors have thought of introducing a mutation into 1oxP sequence to cause insertion reaction, once the gene gene is inserted is inserted into the genome so as not be deleted (not detached from genomic) , Therefore multiple types of variants 1oxP (lox66, lox71, lox511, lox2272) were designed (Figure 1). These variants 1oxP is known (WO01 / 005,987 discloses, JP 2007-100).

In the present invention, it can also be utilized systems called VLOX. The Vlox a VCre-Vlox is a recombinant system derived from Vibrio (Suzuki, E., Nakayama, M.VCre / VloxP and SCre / CloxP: new site-specific recombination systems for genome engineering.Nucleic Acid Res.2011 , 1-11), Vlox43L, etc. Vlox43R, Vlox2272 is available (Figure 1).

1oxP and nucleotide sequence of the mutant 1oxP and Vlox system shown below (Figure 1).

Further, in the present invention, it can be employed Dre / rox system (Figure 2).

The Dre, a D6- site-specific DNA recombinase, an enzyme which recognizes the following sequence rox sites (Sauer, B.and McDermott, Nucic Acid.Res.32: 6086-6095,2004). Recombinant system using this recombinase and rox recognition sequence is referred to as Dre / rox system. This system has been closely related to Cre-lox system recognizes DNA specificity are different.

The nucleotide sequence of the lox and rox shown below (Figure 2).

In the present invention, is intended to establish a mouse with the street human normal liver, further also aims to establish a liver disease model mice. Therefore, in the present invention so that to express the toxin in the cytoplasm of mouse hepatocytes can induce murine hepatocyte death, for gene manipulation in ES cells. Further, since to produce the mice with normal human liver, it is necessary to grow by transplanting human hepatocytes, in ES cells, to replace the mouse growth hormone gene in the growth hormone gene in humans. Furthermore, to analyze the function of such drug metabolism, a mouse drug-metabolizing enzyme gene replaced with drug-metabolizing enzyme gene in humans.

Since mouse introduced hepatic cell death is lost liver function, the mouse except that available as liver injury model, by transplanting human normal liver cells, it is possible to obtain a mouse liver has been humanized.

Figure 3 is a construction diagram of the homologous recombination vector for replacing the mouse growth hormone (GH) gene in the human GH gene.

Further, FIG. 4 is a Cyp gene is a drug metabolizing enzyme gene a construct view of homologous recombination vector for replacing the human Cyp gene.

Substitution of the mouse gene to the human gene can be carried out according to the gene trap method described in Japanese Patent WO01 / 005,987. For example, using the street fabricated vector, performing a two-step gene trap.

The first step is conventional gene trap method. In the normal gene trap, introducing the trap vector into ES cells, to trap the endogenous gene present inherently in the ES cells. Thus, endogenous gene in ES cells are destroyed. Next, connect downstream of lox sequence human genes on a plasmid (replacement vector) (e.g. 1ox66 etc.), for gene trap of the second stage (Fig. 3 and 4).

In the second stage of the gene trap, a human gene linked downstream of 1ox66 (hGH, hCyp etc.) is introduced into ES cells. Thus, 1Ox71 site of the trap vector introduced in the first stage, causing a recombination between lox66 vector introduced in the second stage, "(lox71 / 66) - (human gene) - (loxP) capable of introducing a modified gene that contains a cassette consisting of ". Between the human gene and the loxP may connect the puromycin resistance gene (puro).

According to this method, the endogenous mouse gene can be replaced with human genes. 3 and 4 show views of a replacement allele.

Here, in FIG. 3 and 4, Ex1, Ex2, Ex3 and Ex4 each represent exons 1-4 of mouse growth hormone gene and the mouse Cyp3a13 gene, pA represents a poly A sequence, Frt recognition site of FLP, PGK-neo neomycin resistance gene PGK promoter linked, P-puro represents puromycin resistance gene PGK promoter linked.

5. Fabrication of chimeric mouse chimeric mouse can be performed by standard methods.

First, the established ES cells, or ES cells gene has been introduced or substituted, or to aggregate with 8 cell stage embryos, or injected into blastocyst method. Say Thus the embryos produced a chimeric embryo, but to produce chimeric mice by childbirth by implanting the chimeric embryo into the uterus of a pseudopregnant foster mother.

For example, production of chimeric embryos, first, the female mice were subjected to superovulation treatment by hormonal agent, it is bred with male mice. Then, to recover the initial embryo from oviducts or uterus after a predetermined number of days. The recovered embryos, ES cells were aggregated or injected to produce chimeric embryos.

Here, "embryo" means a stage of an individual to fertility from fertilization in ontogeny, encompasses 2-cell stage embryos, 4-cell embryos, 8-cell embryo, Kuwamikihai, such blastocysts to. 2. Day 5 from fertilization in the case of using the 8-cell stage embryos, in the case of using the blastocysts can be recovered early embryo 3. Day 5 into the oviducts or uterus, respectively fertilization.

As a method of making an aggregate using ES cells and embryos, it is possible to use microinjection, known methods such as aggregation method. The "aggregate" means an aggregate that ES cells and embryonic form gathered into the same space, form ES cells were injected into embryos, give away the embryo into individual cells, aggregate with ES cells It means any of the form.

When employing the microinjection method, the recovered embryos, ES cells injected to prepare a collection of cells. Also, in the case of employing the aggregation method, the ES cells, it is sufficient to agglomerate sprinkled normal embryos remove the zona pellucida.

On the other hand, pseudopregnant female mice for the foster mother may be obtained by mating a female mouse of normal sexual cycle with a male mouse castrated by vasoligation or the like. Relative produced the pseudopregnant mouse, chimeric embryos produced by the method described above was transplanted into the uterus, it is possible to produce chimeric mice by subsequently giving birth.

Among these chimeric mice, selects a male mouse ES cell-derived embryos transferred. After the chimeric mice selected males mature, the mice are bred with female mice of inbred mouse strains. Then, the pups born, by coat color of mice derived from ES cells appear, can be pluripotent stem cells to confirm that it is introduced into the germ line of the chimeric mouse.

6. Preparation of humanized murine (1) Transgenic mice producing gene optimum genetically modified mice humanized were established using introduced or substituted ES cells, genetically modified mice, as described below, 100% human it is the underlying mouse for the establishment of a mouse with a phased liver.

For the avoidance of rejection using ES cells ES cells or BRJ mice NOJ mice.

(I) NOJ (NOD / SCID / Jak3 - / -) mice: C3, T, B, NK, Preparation of NKT deficient Noj mice were mated NOD mice and SCID mice, SCID mutation in the genetic background of NOD to introduce. Further, by mating with Jak3 deficient mice, mice with SCID and Jak3 genetic defects in the genetic background of the NOD (Noj mice) are obtained. In this mouse, complement C3 is deficient, also T cells, B cells, NK cells, NKT cells also mice deficient.

(Ii) BRJ (BALB / c; Rag2 - / -; Jak3 - / -) mice: T, B, NK, in NKT deficient present invention can be used BRJ mouse in addition to the above NOJ mice. BRJ mice are mice that were introduced Rag2 and Jak3 genetic defects in the genetic background of the BALB / c mice, T cells, B cells, NK cells, NKT cells are deficient. Compared to NOJ mice breed easily, it can be produced a number of mice.

(2) Preparation liver damage model mouse liver injury model mice, administration of an antiestrogen, that by expressing the toxin removing mouse hepatocytes (killing), a fault model mice liver function is lost it can be produced.

For killing the mouse hepatocytes, and in order to express the Dre-ER T2 in the cytoplasm of mouse hepatocytes to produce constructs 1 and 2 below. The Dre-ER T2, and Dre recombinase gene, a vector connecting the mutated estrogen receptor gene modified so as not to bind estrogen produced in mammalian body.

Construct 1:

CAG-ATG-rox-EGFP-rox-DT-A

Construct 2:

SAP-Dre-ER T2

Construct 1 is directly below the CAG promoter, which are connected to (i) ATG, EGFP flanked by (ii) rox, and (iii) DT-A (diphtheria toxin fragment A).

Initiation codon and rox upstream of the ATG of EGFP is designed so that the frame fits. Furthermore, the initiation codon of the DT-A is removed, designed to fit the ATG and the frame of the rox upstream.

Construct 2, hepatocyte specific serum amyloid P component: immediately below promoter (serum amyloid P component SAP), which are connected to Dre-ER T2.

By co-introduction of these constructs 1 and 2 in the ES cells of the present invention, it occurs site-specific recombination after tamoxifen can induce hepatocyte specific diphtheria toxin expressed cell death.

That is, as a non-steroidal antiestrogens, such as tamoxifen (Tamoxifen) is the estrogen receptor, competitively binding to estrogen, a substance having anti-tumor activity by exhibiting anti-estrogenic effect. The administration of tamoxifen humanized mice was expressed Dre-ER T2, Dre-ER T2 is translocated to the nucleus by tamoxifen. It occurs recombination between two rox, the promoter of the diphtheria toxin gene to function. Thus, toxin DT-A is expressed, mouse hepatocytes are killed (Figure 5).

Number of doses and timing of administration of tamoxifen is not particularly limited as long as possible kill hepatocytes, for example, as follows.

Tamoxifen ethanol dissolved, the solution was diluted with sun flower oil (S5007, Sigma), adjusted to a concentration of 7 mg / ml. This solution was used to administered in 4 consecutive days intraperitoneally to become adult to 105mg / kg body weight.

(3) mice produced hepatocytes humanized mouse hepatocytes were substituted for human hepatocytes were substituted for human hepatocytes, to remove the mouse hepatocytes by administration of the street antiestrogens, human liver cells by transplanting to the mouse, it is possible to obtain a humanized mouse hepatocytes were substituted for human hepatocytes.

To establish the mice with normal human liver, and the maintenance of liver function over a long period of time, it is necessary to confirm the safety.

(I) mouse growth hormone gene for human liver cells following Preparation transplantation of ES cells which are substituted in the human genes to be able to grow, mouse growth hormone gene replaces the human gene in the ES cell stage.

Specifically, a gene replacement of the street ES cells carried out in two steps.

In a first step, BRJ ES cells (BRJ ES introduced with SAP-Dre-ER T2 and CAG-rox-EGFP-rox- DT-A: SAP-Dre-ER T2; CAG-rox-EGFP-rox-DT perform homologous recombination with the referred to as -A), as well as destruction at the start codon of mouse growth hormone gene, ES cells in this area lox71-PGK-neo-loxP incorporated (BRJ ES :: SAP- Dre-ER T2; CAG-rox -EGFP-rox-DT-a; Gh neo) establishing.

In a second step, using the ES cells with a replacement vector, ES cells human growth hormone gene cDNA is integrated in place of the neo gene (BRJ ES: SAP-Dre-ER T2; CAG-rox-EGFP-rox- DT-a; Gh hGH) can be established.

By using the thus established ES cells, it is possible to obtain a mouse producing human growth hormone.

(Ii) number of doses and timing of administration of removal tamoxifen mouse hepatocytes and undifferentiated hepatocytes are as defined above.

(Iii) human liver cells to produce transplantation of human hepatocytes to be transplanted can be derived from iPS cells.

Human liver cells can be from human iPS cells, to establish an efficient endodermal and hepatic differentiation inducing method using feeder cells or extracellular matrix.

iPS cells, Oct, Sox, Klf, Myc, Nanog, be induced by introducing a gene encoding three to six transcription factors (nuclear reprogramming factor) including family members, such as Lin somatic it (Takahashi, K., et al.Induction of pluripotent stem cells from fibroblast cultures.Nat.Protoc.2,3081-9 (2007); Fusaki N, Ban H, Nishiyama A, Saeki K, Hasegawa M.Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai viru s, an RNA virus that does not integrate into the host genome.Proc Jpn Acad Ser B Phys Biol Sci.2009; 85 (8): 348-62)..

The Oct family member, for example, Oct3 / 4, Oct1A, Oct6, and the like, Oct3 / 4 is preferred.

The sox (related HMG box SRY) family members, for example Sox1, Sox2, Sox3, Sox7, Sox15 and the like, preferably Sox2.

The Klf (Kruppel like factor) family members, for example Klf1, Klf2, Klf4, Klf5 and the like, preferably Klf4.

The Myc family member, c-Myc, N-Myc, such as L-Myc can be mentioned, c-Myc is preferred.

Nanog is highest expressed in the inner cell mass of the blastocyst, a homeobox protein not expressed in differentiated cells.

The Lin family members, Lin28 and the like which is a marker for example undifferentiated human ES cells.

More specifically, as the transcription factor, Oct3 / 4, Sox2, combinations Klf4 and c-Myc are preferred (Takahashi, K.and Yamanaka, S., Cell 126,663-676 (2006)), other also it can be used a combination of Oct3 / 4, the combination of Sox2 and Klf4 or Oct3 / 4, Sox2, Klf4 and L-Myc,.

The somatic cells, such as skin cells, liver cells, fibroblasts, and the like lymphocytes.

Method of gene transfer into somatic cells, for example lipofection, electroporation, microinjection, can be mentioned introduction by viral vectors, and is not particularly limited. Viral vectors, such as retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated virus vectors, and Sendai virus. Commercially available vectors such may be utilized Sendai virus (DNAVEC).

When using vectors, may also be genes introduced as can be expressed, operably linked to regulatory sequences such as promoters and enhancers. The promoter, such as the CMV promoter, RSV promoter, and the like SV40 promoter. These vectors further drug resistance gene (e.g., puromycin resistance gene, neomycin resistance gene, ampicillin resistance gene, hygromycin resistance gene) positive selection marker, such as, negative selection marker (e.g., diphtheria toxin A fragment gene or thymidine such as kinase gene), IRES (internal ribosome entry site), it is possible to include a terminator, such as an origin of replication.

Somatic cells (e.g., 0.5 × 10 4 ~ 5 × 10 6 cells / 100mm dish), at about 37 ° C., under the conditions of MEF feeder or feeder-free, transformer vector comprising said nuclear reprogramming factor When cultured Effects, iPS cells are induced after about 1-4 weeks.

As the medium, for example GMEM medium (Glasgow's Minimal Essential Medium), DMEM (Dulbecco's Modified Eagle's Medium), RPMI1640 medium, and the like OPTI-MEMI medium. The culture medium, KSR (Knockout Serum Replacement), fetal bovine serum (FBS), activin-A, basic fibroblast growth factor (bFGF), retinoic acid, dexamethasone, beta-mercaptoethanol, nonessential amino acids, glutamic acid, sodium pyruvate and antibiotics (e.g., penicillin, etc. streptomycin) selected from such, may be added as appropriate.

After a predetermined period the culture, as in the case of ES cell culture, the cells are harvested by incubating in a medium containing EDTA or collagenase IV. The feeder-free conditions, it is possible to perform cell Matrigel coated plates, in medium conditioned by MEF.

To human hepatocytes from iPS cells, to induce differentiation through three stages are common. In principle,

(A) Induction of the endodermal from pluripotent stem cells,

Induction of (b) within the mesodermal immature liver cells, and (c) the induction of immature liver cells into mature hepatocytes.

activin A and Wnt signaling in the above (a) is thought Hepatocyte growth factor, Oncostatin, Dexamethasone is important in in (b) is FGF or BMP, and (c).

However, the above steps (b) and (c) can be alternatively appropriately DMSO or retinoic acid, FGF4 and hydrocortisone and.

Porting period of human liver cells, embryonic 15.5 days, or adult mice of eight weeks before and after birth.

Transplanted cell number of human hepatocytes is preferably from 10 5 10 6.

Transplantation route of human hepatocytes in the case of embryos implanted by injecting the yolk 嚢静 vein (yolk sac vessel) (Fig. 6,7). For adult, it is injected into the spleen.

(Iv) mouse proliferate mouse growth hormone gene in human hepatocytes were established using the ES cells are replaced with the human growth hormone gene capable of producing human growth hormone. The human growth hormone acts to the transplanted human hepatocytes, encourage their growth, can establish humanized liver mice with human liver normal size.

Confirmation that mouse hepatocytes were replaced in all (100%) human liver cells, namely confirmation of the absence of mouse hepatocytes, the expression of genes expressed in mouse liver by analyzing by RT-PCR method it can be carried out.

(4) The evaluation liver humanized liver mice humanized can be confirmed by examining the following matters either alone or in appropriate combination.

The test item to verify the validation liver function (i) liver function, for example, the following items. Test period is not limited, but is preferably carried out over a year.

Protein-related: total protein, ALB, TTT, ZTT, CRP, Haptoglobin, C3, C4

Non-protein nitrogen component: total bilirubin, direct bilirubin sugars: glucose lipids: triglycerides, total cholesterol, HDL- cholesterol, LDL- cholesterol, ApoAI ApoCII

Enzyme: lactate dehydrogenase (LDH), aminotransferase aspartate (AST (GOT)), alanine aminotransferase (ALT (GPT)), γ- Guru Tamil transferase (GGT), creatine kinase (CK), alkaline phosphatase (AP), amylase (AML)

Others: Calcium, Fe, inorganic phosphate ICG test: administered intravenously to through the indocyanine green (ICG), were measured over time the ICG concentration in blood, examining the livers of dye excretory function. ICG is transported into the liver bound to lipoproteins in the blood, are ingested in hepatocytes during passage through the sinusoids, because they are excreted into the bile without undergoing conjugation, rather than liver cells, the entire liver organ It can be analyzed the function of the.

CT examination: to examine the morphological changes of the liver.

(Ii) using a drug-metabolizing PCR array method, cytochrome P450 analyzing the following drug metabolism-associated enzymes: CYP11A1, CYP11B1, CYP11B2, CYP17A1, CYP19A1, CYP1A1, CYP1A2, CYP1B1, CYP21A2, CYP24A1, CYP26A1, CYP26B1, CYP26C1 , CYP27A1, CYP27B1, CYP2A13, CYP2R1, CYP2S1, CYP2B6, CYP2C18, CYP2C19, CYP2C8, CYP2C9, CYP2D6, CYP2E1, CYP2F1, CYP2W1, CYP3A4, CYP3A43, CYP3A5, CYP3A7, CYP4A11, CYP4A22, CYP4B1, CYP4F11, CYP4F12, CYP4F2, CYP 4F3, CYP4F8, CYP7A1, CYP7B1, CYP8B1.

Alcohol dehydrogenase: ADH1A, ADH1B, ADH1C, ADH4, ADH5, ADH6, ADH7, DHRS2, HSD17B10 (HADH2).

Esterase: AADAC, CEL, ESD, GZMA, GZMB, UCHL1, UCHL3.

Aldehyde dehydrogenase: ALDH1A1, ALDH1A2, ALDH1A3, ALDH1B1, ALDH2, ATDH3A1, ALDH3A2, ALDH3B1, ALDH3B2, ALDH4A1, ALDH5A1, ALDH6A1, ALDH7A1, ALDH8A1, ALDH9A1.

- flavin-containing monooxygenase: FMO1, FMO2, FMO3, FMO4, FMO5.

- monoamine oxygenase: MAOA, MAOB.

- prostaglandin - end Peri oxide synthase: PTGS1, PTGS2.

- xanthine dehydrogenase: XDH.

- dihydropyrimidine dehydrogenase: DPYD.

(Iii) verifying the liver cells in vitro in liver cell function, since it is derived from endoderm, examining temporal expression of genes expressed in endodermal and liver cells, the accumulation of glycogen, the expression like the cytochrome enzyme it is, it is possible to verify whether they have the function of human liver.

Temporal expression of genes expressed in endodermal and liver cells, Oct3 / 4, T, Gsc, Mixl1, Foxa2, Hex, Hnf4a, Hnf6, Afp, Alb, Ttr, can be verified in αAT like. Verification techniques, for example, typical Northern blotting, RT-PCR method, a Western blotting.

Secretory capacity of hepatocytes, can be verified ALB, transferrin, alphal-antitrypsin, a concentration measurement in the culture broth of fibrinogen. The verification method, for example, a general Western blot method or EIA (enzyme-immuno assay) methods.

Accumulation of glycogen, can be verified by the PAS (periodic acid-Schiff) staining. Periodate generates a selectively oxidized to aldehyde glucose residues, discoloration to red-purple by Schiff's reagent.

Expression of cytochrome enzymes can be verified by analysis of at five major CYP3A4, CYP1A2, CYP2C9, CYP2C19 and CYP2D6. Verification techniques, for example, typical Northern blotting, RT-PCR method, a Western blotting.

(5) mice produced the present invention the liver disease model mice substituted with liver cells from a human patient, with transplanted liver cells from a human patient, to remove the murine hepatocytes by administering an antiestrogen it is thus possible to obtain a human liver disease model mice.

To establish a mouse having a human mutation liver is necessary for the establishment and pathological analysis of disease models of the same symptoms as the human patient. The human disease optimization model is established, it can be utilized to develop a versatile new treatments.

Hereinafter, a more detailed description of the present invention through examples. However, the present invention is not limited to these examples. Incidentally, the induction of hepatocytes from iPS cells, a human patient familial amyloid polyneuropathy, establishment of iPS cells from a patient's Hitopuropion acidemia, for transplantation experiments to mice with induced human hepatocytes , ethics Committee, animal Care and Use Committee, apply to the second type of recombinant DNA experiment safety Commission, all been approved.

In Establishment embodiment of ES cells, for the establishment of optimal humanized best mouse human liver cell transplantation, it performs establishment of ES cell lines from BRJ mouse embryos, was also established mouse line.

(1) BALB / c; Rag2 - / -; Jak3 - / - (BRJ) by crossing established Rag2 deficient and Jak3 deficient mice Establishment and ES cell lines in mice were established double deficient mice BRJ (Ono A , et al.J Biomed Biotechnol 2011; 539748,2011.doi: 10.1155 / 2011/5397481)). Using an established BRJ mice subjected to in vitro fertilization, 64 give the embryonic blastocyst, cultured at which conventionally used GMEM-KSR media (14% KSR, 1% FBS, 1000U / ml LIF in GMEM) was carried out, we tried to establish, were only able to establish a very bad two systems of growth.

These were produced chimeric mice used, obtained only about 50% of chimerism, it did not contribute to germlines. Therefore, in addition to the medium PD0325901 of CHIR99021 and MEK inhibitors of inhibitors of GSK3 which are useful for the undifferentiated state maintenance of ES (GMEM-KSR-2i medium), I attempted ES establishment again.

Specifically, it was taken embryos of BRJ in vitro fertilization. Blastocysts 64 was 4 days until blastocysts cultured in KSOM medium, were placed one by one embryo 48-well (gelatin-coated only). Use medium is KSR-GMEM-2i medium. As a medium composition, in the G-MEM (Glasgow minimum essential medium), 1X MEM nonessential amino acids, 0.1mM β-mercaptoethanol, 1mM Sodium pyruvate, 1% Fetal bovine serum (FBS) (Hyclone), 14% Knockout TM SR (KSR), 1100 uints / ml Leukemia inhibitory factor (LIF), contains 2 [mu] M PD0325901 and 3 [mu] M CHIR99021. Culture period is carried out for 14 days, it was changed twice a medium in the middle. To after 18 days from 14 days after, was subcultured from the well that ICM has increased to 24well of feeder cells with. Furthermore, successively, 12-well, 6-well, it performed subcultured into 6-cm dish, and finally, it was possible to completely established without ES strain 28 strains problem with the growth rate and morphology.

(2) BRJ ES cell chimeric mice produced and BRJ that strain using Rag2 - / -; Jak3 - / - using eight cell lines of ES strain was established Establishment of mouse strains, by crossing B6 female and BDF1 male by performing the resultant Morura embryos and aggregation were chimeric mice produced (Table 1).

100% chimeras obtained with three ES lines (BRJ-5, BRJ-6 and BRJ-8), was confirmed germline transmission (Table 2).

It should be noted that, of the obtained ES cells, referred to as the 8 th cell line as "BRJ8", with the date 2012, March 23 (date of receipt), National Institute of Technology and Evaluation Patent Microorganisms Depositary Center (Yubinbango292- was an international deposit under the Budapest Treaty in 0818, Chiba Prefecture Kisarazu Kazusa legs bowed in 2-5-8 NITE Department of biotechnology Patent microorganisms Depositary Center). The receipt number is a "NITE ABP-1297".

To generate a construct specifically kill it can be genetically engineered mice produced hepatocytes for induction of induction (1) mouse hepatocytes death mouse hepatocytes death, to prepare two types of constructs.

Construct 1 (CAG-ATG-rox-EGFP-rox-DT-A) directly below the CAG promoter A promoter, are connected ATG, EGFP flanked by rox, and DT-A a (diphtheria toxin fragment A).

Initiation codon and rox upstream of the ATG of EGFP was designed so that the frame fits. Furthermore, the initiation codon of the DT-A was designed to fit the ATG and the frame of the rox upstream removed.

Construct 2 (SAP-DreER T2) is hepatocyte specific serum amyloid P component: connecting the DreER T2 immediately below promoter (serum amyloid P component SAP). In addition, upstream of the SAP promoter A promoter connecting the puromycin resistance gene.

Specific method is as follows.

(1-1) construct 1

Construct 1, was prepared as follows.

(I) p6SEAZ the PstI, treated with restriction enzymes to pSP-rox2 with KpnI, and blunt-ended with T4 Polymerase (TaKaRa). Thereafter, the restriction enzyme treatment with EcoRI, ligated to prepare a pSP-rox-EGFP-rox.

(Ii) pSP-rox-EGFP-rox and pBSK-atg-rox2 (synthetic DNA, Biomatik Co.) was subjected to restriction enzyme treatment with EcoRI and SmaI, and ligated to prepare a pBSK-atg-rox-EGFP-rox.

The (iii) pBSK-atg-rox-EGFP-rox and P71hAXC-DT performs a restricted with BamHI and PstI, and ligated to prepare a pBSK-atg-rox-EGFP-rox-DT-A.

The (iv) pCAGGS-EGFP with KpnI, the pBSK-atg-rox-EGFP-rox-DT-A was digested with SpeI, and the blunt-ended with T4 Polymerase (TaKaRa). Thereafter, the restriction enzyme treatment with HindIII, and ligated to produce a CAG-atg-rox-EGFP-rox-DT-A.

(1-2) construct 2

Construct 2, it was prepared as follows.

(I) the pkSAP-DrePP as template, from initiation codon to the stop codon before was amplified by PCR. The Reverse Primer was added to BamHIsite.

Were ligated pGEM-T Easy Vector and the PCR products were produced T easy-Dre.

(Ii) the pkSAP-DrePP and T easy-Dre was digested with SalI and EcoRI, and ligated, limit T Easy to prepare a SAP (iii) the T Easy Dre and T easy-SAP with SacII and NotI the enzyme treatment, and ligated to produce a T easy-SAP-Dre.

(Iv) using the T Easy-SAP-Dre and pkSA-CremER T2 PP, it was digested with BamHI and NotI, and ligated to produce a T easy-SAP-DremER T2.

(V) the pkSAP-DrePP and T easy-SAP-DremER T2 was digested with SalI and NotI, and ligated to produce a pKSAP-DreER T2.

A (vi) pKSAP-DreERT2 with SpeI, performs a restriction enzyme treatment the pFPacpaF2 with KpnI, was blunt-ended with T4 polymerase (TaKaRa). Thereafter, the pKSAP-DreER T2 perform restriction enzyme treatment of the SalI, PFPacpaF2 with XhoI, and to prepare Puro-SAP-DreER T2 and ligation.

. (2) upon insertion of the introduced human gene of estrogen receptor gene and diphtheria toxin gene in ES cells was performed examination of conditions for efficient expression (Li, Z.et al, Transgenic Res.20: 191- 200,2011.DOI 10.1007 / s11248-010-9389-22).

The presence or absence of PGK-puromycin cassette and IRES, when any combination is analyzed whether the most expression efficiency.

Using homologous recombination vector, the usual method (Zhao, G., Li, Z., Araki, K., Haruna, K., Yamaguchi, K., Araki, M., Takeya, M., Ando, Y .and Yamamura, K.Inconsistency between hepatic expression and serum concentration of transthyretin in mice humanized at the transthyretin locus.Genes Cells 13:. 1257-1268,2008) was destroyed in advance mouse transthyretin (Ttr) the first exon of the gene by. At this time, the first exon of the ATG is destroyed, lox71-PGK-beta-geo-loxP-poly A-lox2272 is provided the target recombinant clones incorporated into that portion.

Then, to prepare two kinds of replacement vectors. Replacement vector 1 includes a lox66-hTTR cDNA-polyA-Frt-PGK-puro-Frt-loxP. Replacement vector 2 contains a lox66-IRES-hTTR cDNA-polyA-Frt-PGK-puro-Frt-loxP. These replacement vectors to the target recombinant clones were respectively introduced at electroporated with Cre expression vector.

As a result, lox71 / 66-hTTR cDNA-polyA-Frt-PGK-puro-Frt-loxP clones (I (-) P (+) and abbreviation) and lox71 / 66-IRES-hTTR cDNA-polyA-Frt-PGK- puro-Frt-loxP clones were obtained (I (+) P (+) and the abbreviation). While any of the two clones with a PGK-puro, I (-) P (+) has no IRES.

Introducing CAG-FLP these two clones by electroporation, remove the PGK-puro between Frt, I (-) P (-) and I (+) P (-) were prepared to clone.

Generated mice from the four ES clones was subjected to expression analysis, I (-) expression in P (+) is the highest, I (-) P (-), I (+) P (+) , I (+) P (-) expression in the order it was learned that decrease. Further, I (-) in the case of P (+), the expression of human liver TTR (transthyretin) was found to be the expression level of mouse Ttr in control mice (Transthyretin) is substantially the same .

As a result, there is a PGK-puromycin, a combination that does not exist the IRES has revealed that the best expression efficiency of the inserted human gene.

With substituted homologous recombination vector with human growth hormone gene was disrupted in advance mouse growth hormone (Gh) first exon of the gene by conventional methods in the same manner as in Example 2. At this time, the first exon of the ATG is destroyed, lox71-PGK-beta-geo-loxP-poly A-lox2272 is provided the target recombinant clones incorporated into that portion. Then, to prepare a replacement vector. Replacement vectors includes lox66-hGH cDNA-polyA-Frt-PGK-puro-Frt-loxP. The replacement vector to the target recombinant clones were introduced at electroporated with Cre expression vector.

As a result, to obtain a ES clones Gh gene in mice has been replaced with human GH gene.

Using a pre-homologous recombination vector by substitution usual way by human drug-metabolizing enzyme gene, and destroyed the first exon of the mouse Cyp3a13 gene. At this time, the first exon of the ATG is destroyed, lox71-PGK-beta-geo-loxP-poly A-lox2272 is provided the target recombinant clones incorporated into that portion. Then, to prepare a replacement vector. Replacement vectors includes lox66-hCYP3A4 cDNA-polyA-Frt-PGK-puro-Frt-loxP. The replacement vector to the target recombinant clones were introduced at electroporated with Cre expression vector.

As a result, Cyp3al3 gene mice, was obtained ES clones replaced with human CYP3A4 gene.

The method for inducing differentiation of human liver cells from producing human iPS cells of the liver humanized murine almost established, the production of constructs for the induction of mouse hepatocytes death was also performed.

(1) it was constructed from differentiation inducing human iPS cells of liver cells from human iPS cells, efficient endoderm and liver differentiation inducing method using feeder cells or extracellular matrix.

For the evaluation of purification and differentiation induction efficiency of differentiation liver cells, albumin - to establish a reporter gene introduced human iPS cell lines were used to develop differentiation inducing method. For the culture substrate, M15 cells are supporting cells (feeder cells), artificial basement membrane (synthesized Basement Membrane: sBM), using such Cell-ABLE.

To differentiate from the iPS cells into human hepatocytes from day one to day 9 were incubated with 4,500 mg / l glucose DMEM medium. The DMEM medium containing the following: insulin (10mg / l), transferrin (5.5mg / l), sodium selenite (6.7mg / ml), ALBUMAX II (2.5mg / ml), 100mM nonessential amino acids, 2mM L-Glutamine, 50mg / ml streptomycin, 100μM β-mercaptoethanol, activin A (20ng / ml), bFGF (50ng / ml).

Then, from day 9 until day 11 were cultured with retinoic acid (10 -6 M).

Finally, it was differentiated into hepatocytes by culturing transferred to 2,000mg / l glucose DMEM medium until 30 days from the 11th day. The DMEM medium used at this time include the following: 10% KSR, 100mM nonessential amino acids, 2mM L-Glutamine, 50mg / ml streptomycin, 100μM β-mercaptoethanol, Hepatocyte growth facor (10ng / ml), Dexamethasone (1mM), Dimethylsulfoxide (1%), and Nicotinamide (1mM).

(2) a study liver transplantation method iPS from human hepatocytes required to humanize the purpose of establishing a method of introducing efficiently iPS derived hepatocytes mouse liver, iPS from human hepatocytes embryonic vessels present on amnion 17 day fetal mouse (yolk sac vessel) was preliminary experiment method of introducing via (Figure 6,7).

Introduced into a blood vessel that exists (A) dye (blue-dye) and the (B) glycerol micelles encapsulating GFP expression vector to embryonic day 17 of amnion on to confirm the transduction efficiency.

As a result, implant is localized concentrated in the liver, and it was confirmed that dispersed throughout the liver. Also, no effect on survival and delivery of the fetus even after this treatment, (A), efficiently introduced neonatal mice were born in the liver with (B).

The hepatocytes were prepared in the section above (1) was used for transplantation.

Hepatocytes induced to differentiate on the upper and SBM M15 shown in FIG.

Next, in order to induce a large number of liver cells, Cell-able (Toyo Gosei, Tokyo) were cultured using a culture plate that.

As a result, also the 30-day culture, large quantities can be induced to differentiate into hepatocytes, also spheroid formation was performed (Figure 9).

In any method for culturing, endoderm Sox17 positive at day 10 cultures, immature liver cells AFP-positive on day 20 cultures, mature hepatocytes ALBUMIN positive induced differentiation day 30 cultures .

As a function evaluation of differentiation-inducing mature liver cells, the accumulation of glycogen in the PAS staining, was indocyanine green (ICG) staining the detoxification ability.

As a result, liver cells induced to differentiate were found to have the desired function.

Also, liver cells because they do not recognize the expression of the murine gene by RT-PCR analysis using mouse specific primers, it was found that 100% of human origin.

In Establishment embodiment variant humanized liver mice were bred in the mouse model of FAP and PA.

(1) induction of mutations hepatocytes from a human patient (i) familial amyloid polyneuropathy (FAP): Establishment already FAP is transthyretin (transthyretin: TTR) autosomal dominant disease caused by point mutations in the gene it is. For example, the FAP, in the amino acid sequence of transthyretin, 30 th amino acid is substituted with methionine valine (Val30Met). It was established iPS cells using the fibroblast cells taken from patients with this Val30Met mutation.

From this iPS cells, in the same manner as described thus far, it was found to be induced to differentiate into hepatocytes.

(Ii) Hitopuropion establishment PA of iPS cells from patients with acidemia (PA) is propionyl CoA carboxylase (propionyl CoA carboxylase: PCCA) is an autosomal recessive genetic disease caused by abnormal gene. For example, the PA, in the amino acid sequence of PCCA, 52 arginine is replaced with tryptophan (Arg52Trp). Fibroblast cells taken from patients with this mutation was established iPS cells using. From this iPS cells, in the same manner as described thus far, it was found to be induced to differentiate into hepatocytes.

(2) (mice generated by transplanting the hepatocytes derived from iPS from normal human) established the establishment of variant humanized liver mice, humanized liver mice mutated humanized liver mice (a mouse model of FAP and PA) the Like the manufacturing method, the liver cells obtained by differentiation induction from FAP and PA from patients iPS cells can be established by transplanting to the mouse of the present invention.

The present invention, ES cells from immunodeficient mice are provided. By human hepatocytes transplanted into embryos produced using ES cells of the present invention, the liver can produce a humanized mouse, it is possible to examine the human liver function.

Display of microorganisms: "BRJ8"

Receipt number: NITE ABP-1297

The original deposit date (date of receipt): March 23, 2012 international depositary authority: National Institute of Technology and Evaluation, Patent Microorganisms Depositary Center Yubinbango292-0818 Chiba Prefecture Kisarazu Kazusa legs bowed in 2-5-8 NITE Department of Biotechnology Patent microorganisms Depositary

SEQ ID NO: 1 to 15: synthetic DNA

Claims (16)

Rag2 gene and immunodeficient mouse embryos Jak3 gene together deficient embryonic stem cells obtained by culturing in the presence of a GSK3 inhibitor and a MEK inhibitor.

Receipt number is given by NITE ABP-1297, embryonic stem cell of claim 1.

Estrogen receptor gene and diphtheria toxin gene is introduced, embryonic stem cell according to claim 1 or 2.

Growth hormone gene inherent in the cell is replaced with one derived from human embryonic stem cell of claim 3.

Moreover, drug-metabolizing enzyme gene inherent in the cell is replaced with one derived from human embryonic stem cells of claim 4.

Drug metabolizing enzyme gene inherent in cells, Cyp3a11, Cyp3a13, Cyp3a25 and at least one in embryonic stem cell of claim 5 which is selected from the group consisting of Cyp3a41.

Mice generated using embryonic stem cell according to claim 1 or 2.

It was generated using embryonic stem cell according to any one of claims 3-6, transgenic mice.

Cause liver cell damage by administration of an antiestrogen, a mouse according to claim 8.

With transplanting human hepatocytes into mice according to claim 8, mouse, characterized in that the removal of the said mouse-derived hepatocytes by administering an antiestrogen, the liver has been humanized.

Human liver cells, mouse of claim 10 is derived from a patient with liver disease.

A mouse according to claim 11, human liver disease model mice.

The Rag2 gene and Jak3 gene immunodeficient mouse embryos both deficient, characterized by culturing in the presence of a GSK3 inhibitor and a MEK inhibitor, a manufacturing method of embryonic stem cells from immunodeficient mice.

Which comprises administering an antiestrogen in mice according to claim 8, wherein the production of liver damage model mice.

With transplanting human hepatocytes into mice according to claim 8, by administering an anti-estrogen agent and removing a mouse-derived hepatocytes, the method producing mouse liver humanized.

Human liver cells is derived from a patient with liver disease, the method of claim 15.

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4 Sept 2018

Humanized mouse

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