Male embryonic stem cell of BALB / c mouse strain and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a male embryonic stem cell of a BALB / c mouse strain and application of the male embryonic stem cell. The name of the male embryonic stem cell is mouse embryonic stem cell MX4, and the male embryonic stem cell is classified and named as mouse embryonic stem cell, and is preserved in Guangdong Microbial Culture Collection Center on the fifth floor, No.59 building, No.100 Courtyard, Xianlie Middle Road, Guangzhou City on July 10, 2024, and the preservation number is GDMCC No.64854. The method has high tetraploid efficiency (the efficiency of a tetraploid compensation technology), and can be used for preparing mouse chimera embryos or mice.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to male embryonic stem cells of a BALB / c mouse strain and applications thereof. Background Art

[0002] Tetraploidy refers to a cell that contains four times the minimum number of chromosomes, that is, an individual or cell that has four complete sets of chromosomes. Under natural conditions, the incidence of tetraploid mammalian embryos is extremely low and they generally cannot develop normally into a single individual.

[0003] A certain number of embryonic stem cells (ESC) and tetraploid embryos are chimerized to form a chimera. During the development of the chimera, ES cells and tetraploid embryos are not randomly distributed. Tetraploid embryos only participate in the formation of extraembryonic tissues such as yolk sac endoderm and placental trophoblast cells (such as chorionic ectoderm, trophoblast cells, etc.), while ES cells are widely involved in the formation of embryonic bodies, allantoic sacs, amniotic membranes, yolk sac mesoderm, and chorionic mesoderm, but do not participate in the generation of yolk sac endoderm and placental trophoblast cell lineages. That is, the developmental potential of ES cells and tetraploid embryos is complementary, and this phenomenon is called tetraploid complementation technology.

[0004] The animal individuals cloned by this technology are completely developed from ES cells and are called ES animals. The tetraploid compensation technology, which is to inject the ES cell line into the tetraploid embryo carrier in the blastocyst stage, usually has an efficiency of developing ES animals between 1% and 3%. The quality of ESC pluripotency is crucial to the birth efficiency of tetraploids, so it is also very important to screen a high-quality ESC for tetraploids. Therefore, it is necessary to improve the birth rate of chimeric embryonic male ES animals. Summary of the invention

[0005] The first aspect of the present invention aims to provide a male embryonic stem cell.

[0006] The second aspect of the present invention aims to provide a product.

[0007] The third aspect of the present invention aims to provide the use of the male embryonic stem cells of the first aspect of the present invention or the product of the second aspect of the present invention.

[0008] The fourth aspect of the present invention aims to provide a method for preparing mouse chimeric embryos.

[0009] The fifth aspect of the present invention aims to provide a method for preparing mice.

[0010] In order to achieve the above object, the technical solution adopted by the present invention is:

[0011] The first aspect of the present invention provides a male embryonic stem cell, named mouse embryonic stem cell MX4, classified as mouse embryonic stem cell, which was deposited on July 10, 2024 in the Guangdong Provincial Microbiological Culture Collection Center on the 5th floor of Building 59, No. 100 Xianlie Middle Road, Guangzhou, with a deposit number of GDMCC No. 64854.

[0012] In some embodiments, the male embryonic stem cells are derived from Balb / c mice.

[0013] The second aspect of the present invention provides a product comprising the male embryonic stem cells according to the first aspect of the present invention.

[0014] In some embodiments, the product further comprises: mouse 2-cell embryos and electrofusion solution.

[0015] In some embodiments, the electrofusion solution comprises mannitol, MgSO4, CaCl2, and bovine serum albumin.

[0016] In some embodiments, the concentration of mannitol in the electrofusion solution is 0.05-0.4M; further 0.25-0.35M.

[0017] In some embodiments, the concentration of MgSO4 in the electrofusion solution is 0.05-2 mM; further 0.08-0.12 mM.

[0018] In some embodiments, the concentration of CaCl2 in the electrofusion solution is 0.05-2mM; further 0.08-0.12mM.

[0019] In some embodiments, the concentration of bovine serum albumin in the electrofusion solution is 0.01-5 mg / mL; further 2.5-3.5 mg / mL.

[0020] In some embodiments, the product is a kit or a reagent set.

[0021] In some embodiments, the product is used to prepare mouse chimeric embryos or mice.

[0022] In some embodiments, the mouse is at least one of a Balb / c mouse and a CD1 mouse.

[0023] The third aspect of the present invention provides the use of the male embryonic stem cells of the first aspect of the present invention or the product of the second aspect of the present invention in preparing mouse chimera embryos or mice.

[0024] In some embodiments, the mouse is a Balb / c mouse.

[0025] The fourth aspect of the present invention provides a method for preparing mouse chimeric embryos, wherein a mouse tetraploid embryo is polymerized with the male embryonic stem cell of the first aspect of the present invention to obtain a mouse chimeric embryo.

[0026] In some embodiments, the method for preparing the mouse tetraploid embryo is as follows: mixing a mouse 2-cell embryo with an electrofusion solution, performing electrofusion, and obtaining a mouse tetraploid embryo.

[0027] In some embodiments, the mouse 2-cell embryo is the mouse 2-cell embryo in the second aspect of the present invention.

[0028] In some embodiments, the electrofusion fluid is the electrofusion fluid in the second aspect of the present invention.

[0029] In some embodiments, the method for preparing tetraploid mouse embryos further comprises the following step: placing the tetraploid embryo cells in a culture medium for culturing.

[0030] In some embodiments, the culturing time is 8-24 hours.

[0031] In some embodiments, the culture medium is KSOM medium.

[0032] In some embodiments, the culturing further comprises the step of removing the zona pellucida.

[0033] In some embodiments, the mouse is a Balb / c mouse.

[0034] The fifth aspect of the present invention provides a method for preparing mice, wherein the tetraploid mouse embryo obtained by the method of the fourth aspect of the present invention is transplanted into the uterus of a pseudo-pregnant mouse, and the embryo develops to full term pregnancy to obtain a mouse.

[0035] In some embodiments, the mouse tetraploid embryos are cultured overnight prior to transplantation.

[0036] In some embodiments, the mouse is a Balb / c mouse.

[0037] The beneficial effects of the present invention are:

[0038] The present invention provides a male embryonic stem cell, named mouse embryonic stem cell MX4, classified and named mouse embryonic stem cell, which was deposited in Guangdong Microbiological Culture Collection Center on the 5th floor of Building 59, No. 100 Xianlie Middle Road, Guangzhou on July 10, 2024, with a deposit number of GDMCC No. 64854. The embryonic stem cell has a high tetraploid efficiency (the efficiency of tetraploid compensation technology) and can be used to prepare mouse chimera embryos or mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The cell state diagram of male embryonic stem cells (#4) is shown (under a 10x microscope).

[0040] Figure 2 The results of SRY identification of male embryonic stem cells are shown.

[0041] Figure 3 The birth rate of pups produced from male embryonic stem cells is shown. DETAILED DESCRIPTION

[0042] definition

[0043] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0044] As used herein, the term "about" refers to a range of ±20% of the value that follows. In some embodiments, the term "about" refers to a range of ±10% of the value that follows. In some embodiments, the term "about" refers to a range of ±5% of the value that follows.

[0045] As used herein, the term "embryo" is used to refer to the following stages - fertilized oocyte, zygote, 2-cell, 4-cell, 8-cell, 16-cell, morula, blastocyst, expanding blastocyst and hatching blastocyst, and all stages in between (e.g., 2-cell, 3-cell or 5-cell).

[0046] The term "2-cell embryo" as used herein means that after the first cleavage, the embryo contains 2 cells. The embryo at this time is called a 2-cell embryo.

[0047] The term "chimeric embryo" or "chimeric blastocyst" as used herein refers to a blastocyst or embryo in a chimeric state that contains embryonic stem cells. In addition to being produced by an injection method, the chimeric blastocyst or embryo can also be produced using, for example, a so-called "aggregation method" in which embryos + embryos, or embryos + cells, are closely adhered to each other in a covered culture dish to produce a chimeric blastocyst. Furthermore, the relationship between the recipient blastocyst or embryo and the cells to be transplanted in the present invention can be an allogeneic relationship or a xenogeneic relationship.

[0048] The term "blastocyst" as used herein refers to an embryo that has the blastocyst fluid, the blastocyst cavity, inside it.

[0049] The term "embryonic stem cell" or "ES cell" or "ESC" as used herein refers to a cell derived from the inner cell mass of a blastocyst, blastomere or morula that has been serially passaged as a cell line while maintaining an undifferentiated state (e.g., expressing TERT, OCT4 and SSEA and TRA antigens specific to the ES cell of the species). Blastocysts, blastomere, morula, etc. can be derived from in vitro fertilized eggs.

[0050] As used herein, the term "inner cell mass" or "ICM" refers to the mass of cells within the embryo that will eventually give rise to the definitive structure of the embryo.

[0051] The term "tetraploid" as used herein refers to a cell containing four times the minimum number of chromosomes, that is, an individual organism or cell having four complete sets of chromosomes.

[0052] The term "tetraploid compensation technology" used in this article is to polymerize tetraploid (4n) embryos with ES cells to form chimeras. During development, 4n cells mainly participate in the formation of extraembryonic tissues (such as trophoblast, yolk sac membrane, chorioallantoic membrane and placenta, etc.) and hardly participate in the formation of embryos, thereby obtaining animal individuals that are almost completely 2n embryos or ES cells, such as mice.

[0053] The term "transplantation" as used herein refers to the transfer of living tissues or cells from a donor to a recipient, with the intention of maintaining the integrity of the transplanted tissue or cells' functions in the recipient, or enabling the tissue or cells to develop in the recipient.

[0054] As used herein, the term "breeding" refers to the development of an individual animal, such as a mouse.

[0055] The present invention is further described in detail below through specific examples.

[0056] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0057] The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer. The materials and reagents used in the examples are commercially available unless otherwise specified. The manufacturers of the reagents used are indicated, and similar products from other manufacturers are substitutes.

[0058] Example 1

[0059] 1. Isolation and Preparation of Embryonic Stem Cells

[0060] Balb / c mice were mated. 3.5 days after the fertilization of the oocyte, the embryo developed to the blastocyst stage. The vaginal plug of the female mouse was checked every day, and the appearance of the vaginal plug was recorded as day 0 (d0). On d3, the female mouse was killed by cervical dislocation, the abdomen was rinsed with 70% ethanol, and the uterine horns were cut off (cut below the junction of the uterus and fallopian tube and above the cervix). The uterine horns were placed in a sterile Petri Dish and the uterine cavity was rinsed with complete culture medium. The Petri Dish was moved to a dissecting binocular microscope and the blastocyst was found. The blastocyst was collected with a Pasteur pipette. Each embryo was transferred to a 96-well plate covered with a trophoblast and containing ESC culture medium, and the embryo was placed in the center of the well. The culture plate was returned to the incubator and cultured for 4 consecutive days without observation during the period. The embryos were observed after 4 days. On d4-5, the cell clusters from the inner cell mass (ICM) proliferated and gathered together. The culture medium was aspirated from the well plate with embryos and washed with PBS. Then 50μL Accutase was added to digest for 10-15 minutes. During this period, a 50μL pipette was used to repeatedly blow and disperse the cell clumps. After the cell clumps were dispersed under the microscope, ESC culture medium was added to terminate the digestion and the liquid was transferred to a 24-well plate. 500μL fresh ESC culture medium was added for culture. After 3 days, clones were selected for proliferation, passaged and frozen. This operation successfully obtained 10 ESC strains (1#, 2#, 3#, 4#, 5#, 6#, 7#, 8#, 9#, 10#). Among them, the ESC culture medium formula is: Knockout TM DMEM + 10% FBS (Gibco) + Glutamax (Gibco, 100x) + NEAA (non-essential amino acids, Gibco, 100x) + 1 mM β-mercaptoethanol (β-ME) + 10 ng / mL leukemia inhibitory factor (LIF, Millipore) + 1 μM pD0325901 (Selleck) + 3 μM Chir99021 (selleck). The cell status of male ESC (4#) is as follows Figure 1 shown.

[0061] 2. SRY identification

[0062] 2.1ESC genome lysis: 150μL DirectPCR (Tail) added 1.5μL Proteinase K (Proteinase K) to prepare genome lysis solution; pick a single clone, check the survival of the clone in the identification plate under a microscope, aspirate the culture medium in the culture dish, add 12μL lysis solution, use a 100μL pipette to repeatedly blow and transfer the liquid to an eight-tube concatenation, cover the lid, and write the number; put the eight-tube cell lysis sample into the PCR instrument, program: 56℃1.5h, 95℃10min, 16℃ storage. After the program is completed, the cell lysis sample is used as the DNA template for SPY identification.

[0063] 2.2 PCR reaction: Mouse SRY specific primers are shown in Table 1. Different components are added in sequence according to the PCR reaction system in Table 2, and the reaction is carried out in a PCR instrument. The reaction program is: 95℃3min; 95℃15s, 53℃15s, 72℃40s, 35 cycles; 72℃5min; 16℃∞. The SRY identification results are shown in Figure 2 As shown, all 10 ESC lines were male.

[0064] Table 1. SRY primer sequences

[0065]

[0066] Table 2 PCR reaction system

[0067]

[0068]

[0069] 3. Generation of Tetraploid Mice

[0070] 3.1 Inject 7.5 units of PMSG intraperitoneally into 4-10 week old Balb / cF1 female mice. 48 hours later, inject human chorionic gonadotropin (hCG) and cage them with CD1 male mice. The next morning, check the female mice for vaginal plugs and select the female mice with vaginal plugs. Record the corresponding fertilization time of the female mice.

[0071] 3.2 The next day, the pregnant mice were euthanized by cervical dislocation, the abdomen of the mice was disinfected with 70% alcohol, and the abdominal skin and muscle layer were cut open with auxiliary forceps and ophthalmic scissors to open the abdominal cavity. The upper part of one uterine horn was grasped with forceps, and a small incision was made in the membrane near the fallopian tube with scissors, and the connection between the fallopian tube and the ovary was cut off. The fallopian tube and the attached uterus were moved to a 35 mm culture dish, and the fimbria of the fallopian tube was fixed with forceps. A flushing needle filled with M2 culture medium (Hogan, 1994) was gently inserted into the fimbria, and the fallopian tube was flushed with 0.1 mL of M2 culture medium. The embryos flushed out were collected with an oviduct transfer tube and washed with M2 three times to collect the mouse 2-cell embryos of day E1.5 (see 1.5).

[0072] 3.3 The collected mouse embryos were placed in 0.3 M mannitol (Sigma-Aldrich Inc., St. Louis, MO) containing 0.1 mM MgSO4, 0.1 mM CaCl2 and 0.3% (m / v, g / mL) bovine serum albumin, and fused with 60 V 50 microseconds direct current using a Cellfusion CF-150 / B electrofusion instrument and a 250-μm fusion tank (BLS Ltd., Budapest, Hungary) to obtain 4 ploid embryos; the embryos were placed in KSOM culture medium and cultured in a CO2 incubator for 24 hours, and then the zona pellucida was removed with acidic Tyrode's solution (Sigma-Aldrich, T1788), and the embryos were aggregated with the 10 ESCs (1#, 2#, 3#, 4#, 5#, 6#, 7#, 8#, 9#, 10#) isolated as described above to form chimeric embryos.

[0073] The chimeric embryos obtained in 3.4 were cultured in a CO2 incubator overnight and then transplanted into the uterus of a pseudo-pregnant mouse on day E2.5 (see Subsection 2.5). After 17 days, the surrogate mouse was euthanized by cervical dislocation and then subjected to laparotomy. The birth rate of the offspring was counted. The results are as follows: Figure 3 Shown: Chimeric embryos formed from male embryonic stem cells (4#) resulted in a birth rate of approximately 16.6% of offspring.

[0074] The name of male embryonic stem cells (4#) is mouse embryonic stem cell MX4, classified as mouse embryonic stem cell, and was deposited in the Guangdong Provincial Microbiological Culture Collection Center on the 5th floor of Building 59, No. 100 Xianlie Middle Road, Guangzhou on July 10, 2024, with the deposit number GDMCC No. 64854.

[0075] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A male embryonic stem cell, named mouse embryonic stem cell MX4, classified as mouse embryonic stem cell, was deposited in Guangdong Provincial Microbiological Culture Collection Center on the 5th floor of Building 59, No. 100 Xianlie Middle Road, Guangzhou on July 10, 2024, with the deposit number GDMCC No. 64854.

2. A product comprising the male embryonic stem cells according to claim 1.

3. The product according to claim 2, characterized in that The product also includes: mouse 2-cell embryos and electrofusion solution; Preferably, the electrofusion solution comprises mannitol, MgSO4, CaCl2 and bovine serum albumin; Preferably, the concentration of mannitol in the electrofusion solution is 0.05-0.4 M; Preferably, the concentration of MgSO4 in the electrofusion solution is 0.05-2 mM; Preferably, the concentration of CaCl2 in the electrofusion solution is 0.05-2mM; Preferably, the concentration of bovine serum albumin in the electrofusion solution is 0.01-5 mg / mL; Preferably, the product is a kit or a reagent set; Preferably, the product is used to prepare mouse chimeric embryos or mice.

4. Use of the male embryonic stem cell according to claim 1 or the product according to any one of claims 2 to 3 in preparing mouse chimera embryos or mice.

5. A method for preparing mouse chimeric embryos, comprising polymerizing a mouse tetraploid embryo with the male embryonic stem cell according to claim 1 to obtain a mouse chimeric embryo.

6. The method according to claim 5, characterized in that The method for preparing the mouse tetraploid embryo is as follows: mixing the mouse 2-cell embryo with an electrofusion solution, and performing electrofusion to obtain the mouse tetraploid embryo.

7. The method according to claim 6, characterized in that The mouse 2-cell embryo is the mouse 2-cell embryo as claimed in claim 3; and / or The electrofusion fluid is the electrofusion fluid described in claim 3.

8. The method according to any one of claims 5 to 7, characterized in that: The method for preparing tetraploid mouse embryos also comprises the following step: placing the tetraploid embryo cells in a culture medium for culturing.

9. The method according to claim 8, characterized in that The culturing time is 8-24 hours; Preferably, the culture medium is KSOM medium; Preferably, the culturing further comprises a step of removing the zona pellucida.

10. A method for preparing mice, comprising transplanting a tetraploid mouse embryo obtained by the method according to any one of claims 5 to 9 into the uterus of a pseudo-pregnant mouse to obtain a mouse.

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