JP2007181418A - Es cell strain, chimera mouse, knockout mouse, and method for producing them - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an ES (embryonic stem) cell strain suitable for the production of a chimera mouse or a knockout mouse. <P>SOLUTION: The ES cell strain (accession number: FERM AP-20736) derived from a C57BL/6J mouse, enabling the production of the chimera mouse and having differentiation potency to a germ cell is provided. The chimera mouse of a genital system is efficiently and frequently produced by allowing the ES cell to agglutinate with a tetraploid early embryo. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a chimeric mouse comprising at least an embryonic stem cell (hereinafter referred to as “ES cell”) strain derived from a C57BL / 6J mouse, a procedure for agglutinating the ES cell line and a tetraploid early embryo. Alternatively, the present invention relates to methods for producing knockout mice and chimeric mice or knockout mice produced by these methods.

  ES cells are undifferentiated cells derived from the inner cell mass of early embryos, and have the ability to differentiate into all types of cells. Therefore, in recent years, application to regenerative medicine is expected.

  Mouse ES cells were isolated in the 1980s and are now widely applied to the production of knockout mice.

  A knockout mouse is a mouse in which a gene has been deleted or mutated by genetic engineering techniques.

  Knockout mice are generally prepared by the following procedure. First, after preparing a targeting vector (recombinant DNA), the targeting vector is introduced into ES cells by electroporation or the like. Then, ES cell lines that have undergone homologous genetic recombination are selected. Next, the recombinant ES cells are injected into the 8-cell stage embryo or blastocyst stage embryo by the injection method to produce a chimeric embryo. Next, the chimeric embryo is transplanted into the uterus of a pseudopregnant mouse to obtain a litter (chimeric mouse). Next, the produced chimeric mouse and wild type mouse are crossed to confirm whether germ cells are formed by cells derived from recombinant ES cells. Then, mice whose germ cells are confirmed to be formed from cells derived from recombinant ES cells are mated, and knockout mice are selected from the resulting offspring.

In addition, the following documents are mentioned as a prior document relevant to this invention, for example. Patent Document 1 and Patent Document 2 describe a female ES cell line derived from a mouse of the C57BL / 6N strain, a chimeric individual production means using the cell line, and the like. Non-Patent Document 1 describes means for producing ES cell-derived mice by aggregating tetraploid embryos and ES cells as a technique for avoiding embryonic lethality due to gene knockout.
JP 2004-141073 A JP 2001-61470 A Nagy A. et al, “Derivation of completely cell culture-derived mice from early-passage embryonic stem cells” Proc. Natl. Acad. Sci. USA Vol90, pp.8424-8428, September 1993 Developmental Biology

  In general, it is difficult to establish ES cell lines. For example, even when ES cells are prepared according to known procedures, ES cell lines cannot be established in many cases, and even when ES cell lines are established, the ability and tissue to form a chimera for each established strain In many cases, properties and characteristics such as differentiation ability are greatly different.

  In addition, there is a problem that it is difficult to produce a knockout mouse using an ES cell line derived from a mouse of the C57BL / 6 strain. The C57BL / 6 strain mouse is one of the most widely used strains as an inbred mouse, and has an enormous amount of data on traits and attributes. Therefore, it is most preferable to produce knockout mice using the ES cell line of the C57BL / 6 mouse strain. However, when ES cell lines derived from C57BL / 6 strain mice are used, it is difficult to produce germline chimeric mice. Therefore, it is now possible to create germline chimeric mice and establish knockout mice using ES cells derived from 129 mice and ES cells derived from F1 hybrids of C57BL / 6 mice and CBA mice. Many. In that case, it usually takes two or more years to congenic C57BL / 6 strain mice.

  Thus, the present invention establishes an ES cell line suitable for the production of chimeric mice and knockout mice, and uses ES cell lines derived from C57BL / 6 strain mice to efficiently and highly enhance germ line chimeric mice. The main purpose is to provide means that can be manufactured at a high frequency.

  The inventors have succeeded in establishing embryonic stem cells (hereinafter referred to as “ES cells”) derived from C57BL / 6J mice (reception number: FERM AP-20736).

  And as a result of repeated research using the ES cell line, the inventors injected ES cells derived from C57BL / 6 mice into an 8-cell stage embryo or a blastocyst stage embryo by an injection method as before. However, instead of producing a chimeric embryo, it is possible to efficiently produce a germ line chimeric mouse in a short time by aggregating ES cells derived from C57BL / 6 mice and an early tetraploid embryo. Newly found.

  Accordingly, the present invention provides a C57BL / 6J mouse-derived ES cell line (reception number: FERM AP-20736) and a chimeric mouse production method including at least a procedure for agglutinating the ES cells and tetraploid early embryos. To do.

  This ES cell line is derived from C57BL / 6J mice and retains the ability to differentiate into germ cells. Therefore, germline chimeric mice derived from C57BL / 6 mice can be prepared by using this ES cell line. Moreover, chimera mice can be produced with high efficiency and high frequency by aggregating the ES cells and tetraploid early embryos to produce chimera mice. In addition, for example, knockout mice can be established at high frequency by producing germline chimeric mice after genetic recombination of the ES cells.

  When ES cells and tetraploid early embryos are aggregated and then transplanted to mice, the offspring are germline chimeric mice because almost all of the offspring are formed by ES cell-derived cells. It is not necessary to test whether or not by mating. Further, when a knockout mouse having a C57BL / 6 lineage genetic trait is produced, it is not necessary to congenic the mouse of the C57BL / 6 line, so that the production period can be greatly shortened. Therefore, a chimeric mouse or a knockout mouse can be efficiently produced in a short time.

  A tetraploid early embryo can be prepared, for example, by electrically fusing a 2-cell stage embryo of a C57BL / 6 mouse.

  In the present invention, the “germline chimeric mouse” refers to a chimeric mouse that can transmit an ES cell-derived genotype to offspring by mating. That is, it refers to a chimeric mouse in which germ cells (sperm or egg) are formed by cells derived from ES cells, and the genetic information of ES cells can be transmitted to the next generation through reproduction. For example, when an embryonic cell is injected into an early embryo by an injection method to produce a chimeric embryo and a chimeric mouse is obtained, germ cells are formed by cells derived from early embryos, and germ cells are produced by cells derived from ES cells. May be formed. On the other hand, when the germ cells of a chimeric mouse are formed by ES cell-derived cells, the ES cell-derived genotype can be transmitted to offspring by mating using the chimeric mouse.

  According to the present invention, germline chimeric mice can be produced efficiently and frequently.

In Example 1, an ES cell line was uniquely established from an early mouse embryo of the C57BL / 6J strain, and an attempt was made to produce a chimeric mouse using the ES cell line.
The outline is shown below.

  First, an ES cell line was established. After collecting an inner cell mass from a blastocyst of a mouse of C57BL / 6J strain, the collected cell was cultured on a feeder cell to establish a male ES cell derived from C57BL / 6J (reception number: FERM AP- 20736). As feeder cells, BALB / c strain-derived mouse fetal fibroblasts were used after mitomycin C treatment. Further, as a medium, DMEM (DULBECCO's modified EAGLE's medium; Dulbecco's modified Eagle medium, manufactured by SIGMA), 15% KSR (KNOCKOUT serum replacement; manufactured by GIBCO), LIF (Leuk) Inhibitory factor; leukemia inhibitory factor, manufactured by Invitrogen), NEAA (non essential amino acid; non-essential amino acid, manufactured by Invitrogen), and sodium pyruvate (manufactured by Invitrogen) were used.

  Subsequently, an attempt was made to produce a chimeric mouse by the injection method using the established ES cell line. First, 8-cell embryos or blastocyst stage embryos were collected from female mice of the ICR line that were mated. Next, the established ES cells were injected into the embryo by an injection method to produce a chimeric embryo. Next, the chimeric embryo was transplanted into the uterus of a pseudopregnant female mouse, and a litter (male mouse) was obtained by spontaneous delivery. Then, whether or not the obtained male mouse was a mouse having ES cell-derived cells was determined by the hair color. The C57BL / 6J mouse has a black hair color, the ICR mouse has a white color (albino), and the chimeric mouse has a white pattern and a black (or brown) pattern.

  Next, the obtained male chimeric mouse and female mouse of ICR strain were mated, and it was examined whether or not the produced male chimeric mouse was a germline chimeric mouse. When germ cells (sperm) of male chimeric mice are formed by ES cell-derived cells, offspring (male or female mice) obtained after mating are black because they have C57BL / 6J-derived traits. . On the other hand, when germ cells of male chimeric mice are formed of embryonic cell-derived cells, offspring (male or female mice) obtained after mating are derived from ICR, and thus become white hair color. Then, it was investigated whether the produced male chimera mouse was a germline chimera mouse by confirming the hair color of the offspring (male or female mouse) after mating.

  The results are shown in Table 1. In the table, “donor” indicates the ES cell used, “recipient” indicates the origin (strains) and type of embryo (embryo) of the embryo used, and “the techniques used” indicates the introduction of ES cells. Means are shown. In the table, “No. of embryos transferred” indicates the number of chimeric embryos transplanted into pseudopregnant mice, “No. of newborns” indicates the number of offspring born, “No. of chimeras” indicates the number of offspring born. Among them, the number of chimeric mice (number including both males and females) and “No. of germline chimeras” indicate the number of germline chimeric mice among the obtained chimeric mice. In the table, “B6ES” is the result using ES cells established from the early mouse embryo of the C57BL / 6J strain, and “clone A” and “clone B” indicate that the clones are different. “ICR” used an early embryo of a mouse of the ICR strain, “8-cell” used an 8-cell stage embryo, “blastocyst” used a blastocyst stage embryo, “injection” Represents that ES cells were injected by the injection method.

  FIG. 1 is a drawing-substituting photograph of the produced chimeric mouse. In the two mice on the right, there were few black parts and the occupancy rate of cells derived from ES cells was low. Subsequently, as a result of mating female mice of the ICR strain, the ability of germline inheritance could not be confirmed (somatic chimera). On the other hand, in the two mice on the left, the occupation rate of cells derived from ES cells was close to 100%, and the whole body was black. Thereafter, as a result of mating female mice of the ICR strain, it was confirmed that they were germline chimeric mice (germline chimera).

  As shown in Table 1 and FIG. 1, in this experiment, a male ES cell line can be established from an early mouse embryo of the C57BL / 6J strain, and the ES cell line is also used by an injection method which is a general method. Male germline chimeric mice could be generated.

  This experimental result is advantageous in that an ES cell line can be established from an early mouse embryo of the C57BL / 6J strain and a germ line chimeric mouse can be produced using the ES cell line. is there. There are several genetically distinct sublines in the C57BL / 6 strain of mice. For example, 6J is a sub-system established at the Jackson Laboratory in the United States, and 6N is a sub-system established at the National Institute of Health (NIH) in the United States. Among them, the 6J line is the most widely used line as a prototype (standard line) in studies such as mouse genome analysis. Therefore, this experiment is advantageous in that a chimeric mouse that can be transferred to the germline can be produced using the ES cell line derived from the 6J strain of the C57BL / 6 strain.

  In addition, the above experimental results are advantageous in that a male ES cell line can be established and a male germline chimeric mouse can be produced using the ES cell line. . When male ES cells are used, male germline chimeric mice can be obtained. Therefore, by using the sperm, many offspring mice can be obtained at one time. Therefore, ES cell-derived mice can be produced efficiently. In addition, since male ES cells have both the X chromosome and the Y chromosome, there is an advantage that a gene existing on the Y chromosome can be analyzed.

  In Example 2, an attempt was made to produce a chimeric mouse using an ES cell line and a tetraploid embryo.

  First, tetraploid embryos were prepared. Two-cell embryos were collected from C57BL / 6J strain mice and tetraploidized by electrofusion. Next, this tetraploid embryo was cultured to the 8-cell stage. Next, after removing the tetraploid embryos, ES cells derived from the C57BL / 6J strain were added, and both were aggregated. In this experiment, instead of the ES cells established in Example 1, ES cells derived from C57BL / 6J-green mouse (transgenic mouse introduced with GFP gene) separately established by the inventors were used. Since this ES cell and the ES cell established in Example 1 have the same mouse strain, it can be assumed that the genetic traits are the same except that the GFP gene is introduced. Next, the aggregated embryos were developed into blastocysts and then transplanted into pseudopregnant mice to obtain offspring (male mice).

  In a general electrofusion method, a plurality of two-cell stage embryos are arranged in a straight line, and both sides are sandwiched between parallel double electrodes to perform electrofusion. However, when electrofusion is performed by this method, cell fusion cannot be uniformly performed with all cells, and a tetraploid embryo cannot be reliably produced. Therefore, since the diploid embryo is mixed in the produced tetraploid embryo, there is a problem that the incidence of the target mouse is lowered. Therefore, after trial and error by the inventors, in this experiment, electrofusion was performed by sandwiching one 2-cell stage embryo with a needle-type electrode, and tetraploid embryos were produced one by one. As a result, the production efficiency of the tetraploid embryo was lowered, but a tetraploid embryo could be reliably obtained.

  The results are shown in Table 2. Each description in the table is the same as in Table 1. In the table, “B6G-2 cells” is a result of using ES cells established from C57BL / 6J-green mouse, and “C57BL / 6J” is an early embryo of a mouse of C57BL / 6J strain. “Tetratrap 8-cell” indicates that tetraploid 8-cell embryos were used, and “aggregation” indicates that tetraploid embryos and ES cells were aggregated by the aggregation method. . Each description in the table is the same as that in Table 1.

  As shown in Table 2, male chimeric mice could be produced at high frequency by using a method of aggregating ES cell lines derived from C57BL / 6J strain mice and tetraploid embryos.

  This method for producing a chimeric mouse is advantageous in that it is not necessary to separately mate and examine whether it is a germline chimeric mouse. When this method is used, tetraploid cells die during development, and almost all cells of the produced male chimeric mouse are formed from ES cell-derived cells. Therefore, since germ cells are also formed from cells derived from ES cells, it is not necessary to separately examine whether they are germ line chimeric mice by mating or the like.

  In addition, the above-described method for producing a chimeric mouse is advantageous in that it does not require separate mating in order to produce a mouse formed only from cells derived from ES cells. When producing a chimeric mouse by the injection method, for example, after crossing the produced chimeric mouse with a wild type mouse and confirming whether it is a germ cell chimeric mouse, the resulting offspring are further mated with each other, and ES A mouse formed only with cell-derived cells is produced. On the other hand, when producing chimeric mice according to the above-described procedure, it is not necessary to cross them.

  Therefore, by using this chimeric mouse production method, a mouse formed only with cells derived from ES cells can be produced in a short period of time, and the production efficiency of those mice can be greatly improved.

  In Example 3, using the ES cell line established in Example 1, an attempt was made to produce a knockout mouse. In this experiment, as an example, an attempt was made to create an angiotensinogen gene-deficient mouse.

  First, the endogenous angiotensinogen gene was recombined to produce recombinant ES cells. First, a targeting vector was prepared. After preparing a DNA having a partial sequence of an angiotensinogen gene, a neomycin resistance gene (neo ') as a positive selection marker and a diphtheria toxin A fragment (DT) gene as a negative selection marker are linked to the DNA, A target vector was prepared. Next, the targeting vector was introduced into ES cells by electroporation. Thereby, since homologous recombination occurs between the endogenous angiotensinogen gene and the targeting vector, a recombinant ES cell can be prepared (see FIG. 2).

  In addition, since the neomycin resistance gene is incorporated into the recombinant ES cells, the recombinant ES cells can be selected by culturing in a medium containing G418 (selection drug). Further, since the neomycin resistance gene is incorporated in the middle of the angiotensinogen gene, the function of the angiotensinogen gene can be deleted.

Table 3 shows the recombination frequency of the angiotensinogen gene in ES cells.
In the table, “TT2” indicates the frequency of gene recombination when a TT2 cell line is used as an ES cell (control). The TT2 cell line is an ES cell line derived from an ES cell collected from an F1 hybrid of a C57BL / 6 mouse and a CBA mouse.
In the table, “B6-ES” indicates the frequency of genetic recombination in ES cells derived from C57BL / 6J strain mice established in this experiment.
In the table, “Number of G418-resistant clones” indicates the number of clones grown when cultured in a G418-containing medium, and “Number of genetically modified clones” is the number of G418-resistant clones by PCR. The number of clones in which genetic recombination was actually confirmed is shown.

  Subsequently, chimeric mice were produced by the same method (injection method) as in Example 1 using the produced recombinant ES cells.

  The results are shown in Table 4 and FIG. FIG. 3 is a drawing-substituting photograph of a chimeric mouse prepared using recombinant ES cells. This chimeric mouse could not be confirmed to be a germline chimera.

  Each description in the table is the same as in Table 1 or Table 2. In addition, “Agt-targeted B6ES cells” is a result of using genetically modified ES cells obtained by performing genetic recombination (knockout) of the angiotensinogen gene on ES cells established from early mouse embryos of the C57BL / 6J strain. Indicates that there is.

  As shown in Table 4 and the like, when chimeric mice (knockout mice) were produced by the injection method using recombinant ES cells, only one chimeric mouse could be produced as a whole. Further, although this male chimeric mouse and a female mouse of the ICR strain were mated, a mouse formed only with cells derived from recombinant ES cells could not be produced.

  Therefore, this time, an attempt was made to produce a knockout mouse by the method of Example 2 (a method using a tetraploid embryo).

  The results are shown in Table 5. Each description in the table is the same as that in Table 1, Table 2, or Table 4.

  As shown in Table 5, male knockout mice could be produced at high frequency by using a method of aggregating recombinant ES cell lines derived from C57BL / 6J strain mice and tetraploid embryos.

  In addition, this method for producing a knockout mouse is that it is not necessary to separately cross-check whether it is a germline chimeric mouse during the procedure, and a mouse that is formed only from ES cell-derived cells is produced. Therefore, there is an advantage in that it is not necessary to perform the mating separately. Therefore, by using this knockout mouse production method, a knockout mouse formed only from ES cell-derived cells can be produced in a short period of time, and the production efficiency of the knockout mouse can be greatly improved.

  In addition, a part of the tail of the produced knockout mouse was cut and a tissue was collected, and whether or not the mouse had a mutated gene (neomycin resistance gene) was examined by a PCR method. The results are shown in FIG.

  FIG. 4 is a drawing-substituting photograph showing the results of PCR. Each band in the figure indicates the presence or absence of a neomycin resistance gene in the fetus.

  Lanes 1 to 5 show the results of PCR in the prepared knockout mice. Lane 1 is the result of an individual who died in the fetus, and lanes 2 to 5 are the result of the surviving fetus. Lane 6 is a negative control, and shows the results of PCR in the case of using tails collected from C57BL / 6J individuals that were not genetically modified. Lanes 7 and 8 are positive controls, and show the results of PCR when tails collected from knockout mouse individuals already incorporating a neomycin resistance gene were used.

  The result of FIG. 4 shows that the gene of interest is knocked out in the produced knockout mouse.

  According to the present invention, germline chimeric mice or knockout mice consisting of only ES cells derived from C57BL / 6J strain mice can be produced frequently in a short period of time.

  As described above, the C57BL / 6 mouse is the most widely used mouse as an inbred mouse, and has an enormous amount of data related to traits and attributes. According to the present invention, a knockout mouse of the C57BL / 6J strain can be prepared. For example, detailed functional analysis of the knocked out gene is performed by comparing and examining data obtained from the knockout mouse and accumulated data. be able to.

The drawing substitute photograph of the chimera mouse produced by the injection method in Example 1. In Example 3, the schematic diagram which shows the homologous recombination between an endogenous angiotensinogen gene and a targeting vector. In FIG. 3, the drawing substitute photograph of the knockout mouse produced using the recombinant ES cell by the injection method. The drawing substitute photograph which shows the result of PCR regarding whether the target gene is knocked out.

Claims (8)

  Embryonic stem cell (hereinafter referred to as “ES cell”) strain derived from C57BL / 6J mouse (reception number: FERM AP-20736).   The ES cell line according to claim 1, wherein a chimeric mouse can be produced, and the ability to differentiate into a germ cell is maintained.   A method for producing a chimeric mouse comprising at least a procedure for aggregating the ES cell line according to claim 1 or claim 2 and a tetraploid early embryo.   The method for producing a chimeric mouse according to claim 3, wherein the tetraploid early embryo is produced by electrically fusing a 2-cell stage embryo of a C57BL / 6 mouse.   The method for producing a chimeric mouse according to claim 3 or 4, wherein the chimeric mouse is a germline chimeric mouse. A procedure for carrying out genetic recombination of the ES cell line according to claim 1 or claim 2,
A method for producing a knockout mouse, comprising at least a procedure for agglutinating the produced recombinant ES cells and a tetraploid early embryo.   A chimeric mouse produced by the method according to any one of claims 3 to 5. A knockout mouse produced by the method according to claim 6.