Preparation method of zebra fish haploid cells

Abstract

The invention provides a preparation method of a zebra fish haploid cell line. The preparation method comprises the following steps: 1) inactivating sperm genetic materials; 2) activating the ova by sperms with inactivated genetic materials, and developing to form haploid embryos; (3) separating and culturing cells in the blastocyst period by using a culture medium suitable for preparing a zebra fish haploid cell line; and 4) establishing a stable zebra fish haploid cell line through passage. The invention also provides the composition of the culture medium suitable for preparing the zebrafish haploid cell line.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and more specifically, relates to a method for preparing zebrafish haploid cells. Background Art

[0002] The study of gene function often involves genetic screening, which is divided into forward and reverse genetic screening. Genetic screening first requires the establishment of a mutant library. Since animal individuals are usually diploid, the cell lines derived and established are also diploid cell lines. Mutations in diploid cells are often heterozygous. Except for chromosomes X and Y, other chromosomes have homologous chromosomes that can make up for the defects caused by one chromosome, so it is difficult to screen cells with phenotypes. The use of haploid cells can solve this problem, because each haploid cell has only one set of chromosomes, and the mutants produced are homozygous mutations, so cells with the expected phenotype can be efficiently screened.

[0003] Haploid cells are a very important tool for genome-wide recessive gene genetic screening. Scientists have used haploid cells to screen a variety of key factors. For example, using the retrovirus mutation library of mouse haploid embryonic stem cells, cell lines with mutations in Rarg (retinoic acid receptor gamma) and Drosha were identified, confirming that these two genes have important functions in embryonic stem cells. Xist is a non-coding RNA that can silence the expression of one of the two X chromosomes. Using forward genetics, Spen (RNA binding protein), a key molecule for Xist's function, was found in mouse haploid embryonic stem cells. The mutant library generated by piggyBac transposon-mediated screening was used to screen key factors related to the DNA mismatch repair mechanism. In addition to viral infection and transposon systems, chemical mutagens can also be used to generate mutant libraries, such as DNA-alkylating agent ethyl methanesulfonate (EMS), for genetic screening of haploid cells. In non-human primates, genetic screening for resistance genes to A803467 (tetrodotoxin) was performed using an established rhesus macaque haploid neural precursor cell line, and it was found that the toxin caused cell death through B4GALT6.

[0004] Haploid cells have become an effective genetic screening tool, providing broad application prospects for the research of clinical drugs, toxins, pathogen mechanisms, stem cells, embryonic development, cell signaling pathways, etc. However, scientists have established haploid cell lines of many species, such as humans, monkeys, mice, rats, frogs, medaka fish, etc., but so far, no zebrafish haploid cell line has been successfully established.

[0005] Therefore, there is an urgent need to fill the gap in the lack of haploid cell lines in zebrafish research and provide effective tools for genetic screening in zebrafish research. Summary of the invention

[0006] The object of the present invention is to provide a method for preparing a zebrafish haploid cell line, comprising: 1) inactivating sperm genetic material; 2) activating eggs by sperm with inactivated genetic material to develop into haploid embryos; 3) using a culture medium suitable for preparing a zebrafish haploid cell line, isolating and culturing cells in the blastocyst stage; and 4) establishing a stable zebrafish haploid cell line by passage. The present invention also provides the composition of a culture medium suitable for preparing a zebrafish haploid cell line.

[0007] In a first aspect of the present invention, a method for preparing a zebrafish haploid cell line is provided, comprising:

[0008] 1) Inactivation of sperm genetic material;

[0009] 2) The egg is activated by the genetically inactivated sperm and develops into a haploid embryo;

[0010] 3) isolating and culturing cells at the blastocyst stage using a culture medium suitable for preparing a zebrafish haploid cell line; and

[0011] 4) Establish a stable zebrafish haploid cell line by subculturing.

[0012] In one or more embodiments, the culture medium suitable for preparing a zebrafish haploid cell line comprises a basal culture medium and additional factors: ZEE, basic FGF, Y-27632, and BMP4.

[0013] In one or more embodiments, the content (volume percentage) of the added factor is calculated based on the total volume of the culture medium:

[0014] ZEE: 1% to 10%; preferably 2% to 8%, 3% to 6% or 4% to 5% (e.g. 5%);

[0015] Basic FGF: 0.001% to 0.1%; preferably 0.001% to 0.05%, 0.01% to 0.05% or 0.005% to 0.2% (e.g. 0.01%);

[0016] Y-27632: 0.01% to 1%; preferably 0.01% to 0.5%, 0.05% to 0.3% or 0.05% to 0.2% (e.g. 0.1%);

[0017] BMP4: 0.001% to 0.1%; preferably 0.001% to 0.05%, 0.005% to 0.05% or 0.005% to 0.2% (eg 0.01%).

[0018] In one or more embodiments, the culture medium further comprises: N2 supplement, B27 supplement, NEAA, 2-Mercaptoethanol, and L-glutamine.

[0019] In one or more embodiments, the N2 supplement content is 0.1% to 5%; more preferably, 0.1% to 3%, 0.5% to 2% or 0.5% to 1.5% (eg, 1%).

[0020] In one or more embodiments, the content of B27 supplement is 0.1% to 5%; more preferably 0.1% to 3%, 0.5% to 2% or 0.5% to 1.5% (eg, 2%).

[0021] In one or more embodiments, the NEAA content is 0.1% to 5%; more preferably 0.1% to 3%, 0.5% to 2% or 0.5% to 1.5% (eg, 1%).

[0022] In one or more embodiments, the content of 2-Mercaptoethanol is 0.01% to 1%; more preferably 0.01% to 0.5%, 0.05% to 0.3% or 0.05% to 0.2% (eg, 0.1%).

[0023] In one or more embodiments, the L-glutamine content is 0.05-2.5 g / L; more preferably 0.05-1.5 g / L, 0.25-1 g / L, 0.25-0.75 g / L or 0.25-0.5 g / L (eg, 0.292 g / L).

[0024] In one or more embodiments, the culture medium further includes: a serum substitute or serum; preferably a serum substitute; preferably, the concentration thereof is ≥5%, preferably ≥10%, and more preferably 5% to 20% (e.g., 5% to 15%, 10% to 20%, 15% to 20% or 10% to 15%) based on the total volume of the culture medium.

[0025] In one or more embodiments, the culture medium also includes an antibiotic; preferably, it is an effective amount of penicillin and streptomycin, and more preferably a penicillin and streptomycin mixture containing glutamine (preferably, the effective amount of penicillin is 0.001% to 0.1%, more preferably 0.001% to 0.05%, 0.001% to 0.02% or 0.005% to 0.015%, for example 0.006%; wherein the effective amount of streptomycin is 0.001% to 0.1%, more preferably 0.001% to 0.05%, 0.001% to 0.02% or 0.005% to 0.015%, for example 0.01%).

[0026] In one or more embodiments, the basal culture medium includes a basal culture medium selected from the following group: DMEM, MEM, DMEM / F12, Advanced DMEM / F12, RPMI; preferably, the culture medium includes a serum substitute, and the basal culture medium is a culture medium combined with a serum substitute (such as KO DMEM).

[0027] In one or more embodiments, the zebrafish is a zebrafish of the ziwi:eGFP or kdrl:mcherry strain.

[0028] In one or more embodiments, in step 1), the inactivation of sperm genetic material is induced by Coy rays, ultraviolet rays, and chemicals (preferably, the chemicals include: toluidine blue, ethylene urea, dimethyl sulfate, acriflavine, thiazide). Preferably, the inactivation of sperm genetic material is induced by ultraviolet irradiation for 5 to 20 seconds (e.g., 5 to 15 seconds).

[0029] In one or more embodiments, in step 3), the blastocyst stage refers to the stage after 4 hours of development after the start of fertilization, and the embryo has a multicellular morphology.

[0030] In one or more embodiments, in step 4), a stable zebrafish haploid cell line is obtained after 10 generations of culture.

[0031] In a second aspect of the present invention, a zebrafish haploid cell line is provided, which is prepared by the method described in the present invention.

[0032] In the third aspect of the present invention, a composition (preferably a culture medium composition) suitable for preparing, isolating, culturing and / or propagating a zebrafish haploid cell line is provided, comprising: a basal culture medium and additional factors: ZEE, basicFGF, Y-27632, BMP4.

[0033] In one or more embodiments, based on the total volume of the composition, the content (volume percentage) of the added factor is:

[0034] ZEE: 1% to 10%; preferably 2% to 8%, 3% to 6% or 4% to 5% (e.g. 5%);

[0035] Basic FGF: 0.001% to 0.1%; preferably 0.001% to 0.05%, 0.01% to 0.05% or 0.005% to 0.2% (e.g. 0.01%);

[0036] Y-27632: 0.01% to 1%; preferably 0.01% to 0.5%, 0.05% to 0.3% or 0.05% to 0.2% (e.g. 0.1%);

[0037] BMP4: 0.001% to 0.1%; preferably 0.001% to 0.05%, 0.005% to 0.05% or 0.005% to 0.2% (eg 0.01%).

[0038] In one or more embodiments, the composition further comprises: N2 supplement, B27 supplement, NEAA, 2-Mercaptoethanol, and L-glutamine.

[0039] In one or more embodiments, the N2 supplement content is 0.1% to 5%; more preferably, 0.1% to 3%, 0.5% to 2% or 0.5% to 1.5% (eg, 1%).

[0040] In one or more embodiments, the content of B27 supplement is 0.1% to 5%; more preferably 0.1% to 3%, 0.5% to 2% or 0.5% to 1.5% (eg, 2%).

[0041] In one or more embodiments, the NEAA content is 0.1% to 5%; more preferably 0.1% to 3%, 0.5% to 2% or 0.5% to 1.5% (eg, 1%).

[0042] In one or more embodiments, the content of 2-Mercaptoethanol is 0.01% to 1%; more preferably 0.01% to 0.5%, 0.05% to 0.3% or 0.05% to 0.2% (eg, 0.1%).

[0043] In one or more embodiments, the L-glutamine content is 0.05-2.5 g / L; more preferably 0.05-1.5 g / L, 0.25-1 g / L, 0.25-0.75 g / L or 0.25-0.5 g / L (eg, 0.292 g / L).

[0044] In one or more embodiments, the composition further includes: a serum substitute or serum; preferably a serum substitute; preferably, the concentration thereof is ≥5%, preferably ≥10%, and more preferably 5% to 20% (e.g., 5% to 15%, 10% to 20%, 15% to 20%, or 10% to 15%) based on the total volume of the culture medium.

[0045] In one or more embodiments, the composition also includes an antibiotic; preferably, it is an effective amount of penicillin and streptomycin, and more preferably a penicillin and streptomycin mixture containing glutamine (preferably, the effective amount of penicillin is 0.001% to 0.1%, more preferably 0.001% to 0.05%, 0.001% to 0.02% or 0.005% to 0.015%, for example 0.006%; wherein the effective amount of streptomycin is 0.001% to 0.1%, more preferably 0.001% to 0.05%, 0.001% to 0.02% or 0.005% to 0.015%, for example 0.01%).

[0046] In a fourth aspect of the present invention, a use of a composition is provided for preparing, isolating, culturing and / or propagating a zebrafish haploid cell line; wherein the composition is the composition described in the present invention.

[0047] In a fifth aspect of the present invention, a kit is provided, wherein the kit comprises the composition of the present invention, or comprises a culture medium suitable for preparing a zebrafish haploid cell line as defined in the present invention.

[0048] In a sixth aspect of the present invention, there is provided a use of the zebrafish haploid cell line of the present invention for genetic screening, gene knockout / knock-in, and preparation of semi-cloned fish.

[0049] Other aspects of the present invention will be apparent to those skilled in the art in view of the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 The left picture shows the cell morphology of medaka fish haploid cell line (Yi M., Hong N., and Hong Y., Generation of medaka fish haploid embryonic stem cells. Science, 2009. 326(5951): p. 430-3.) at different culture days. The scale bar is 20 μm, where ni represents nucleolus and nu represents nucleus. The right picture shows the cell morphology of zebrafish haploid cell line at 3-5 days of culture. The scale bar is 100 μm.

[0051] Figure 2, Schematic diagram of the process of establishing zebrafish haploid cell lines.

[0052] Figure 3 , zebrafish haploid embryos. The embryos in the upper row are normal diploid embryos, and the embryos in the lower row are zebrafish haploid embryos.

[0053] Figure 4 , Establishment of zebrafish haploid cell lines;

[0054] (A) The morphology of zebrafish blastocyst embryos and the morphology of cells isolated and cultured at 0h, 6h, and 24h. The scale bar is 100μm.

[0055] (B) Cell morphology of the zebrafish haploid cell line at passage 60 (P60);

[0056] (C) Flow cytometry analysis of the ploidy of the zebrafish haploid cell line at passage 57 (P57). Control is the control group. The scale bar is 100 μm.

[0057] Figure 5 , identification of zebrafish haploid cell lines;

[0058] (A) Karyotype analysis of haploid cells showed that zebrafish haploid cells contain 25 chromosomes;

[0059] (B) Statistical results of karyotype analysis of haploid zebrafish cells at generation 28;

[0060] (C) Genome stability (CNV) analysis comparing passage 25 (P25) and passage 57 (P47) zebrafish haploid cell lines with the commercial ZF4 cell line. Red indicates increased copy number and blue indicates decreased copy number.

[0061] Figure 6 , the top 10 genes with the highest expression in the zebrafish haploid embryonic cell line. The vertical axis is FPKM, and the horizontal axis is the gene name.

[0062] Figure 7 , the effect of different concentrations (0%, 1%, 2%, 5%) of embryo extract ZEE on the proliferation of zebrafish haploid embryonic cells. The horizontal axis is the cell culture time, and the vertical axis is the relative number of zebrafish haploid embryonic cells (in OD 450 express).

[0063] Figure 8 , the optimization results of the factors maintaining ploidy of haploid cells in zebrafish;

[0064] (A) Cell culture medium supplemented with BMP4 (CM_BMP4) promotes the maintenance of haploidy;

[0065] (B) Cultivation of haploid cells of different zebrafish strains (AB, ziwi:eGFP, sox17:eGFP, kdrl:mcherry) at the 5th generation, Tg indicates transgenic strain;

[0066] (C) The proportion of haploid cells in the G0 / G1 phase at the 5th generation in different zebrafish strains (AB, ziwi:eGFP, sox17:eGFP, kdrl:mcherry). DETAILED DESCRIPTION

[0067] After in-depth research, the inventors provide a method for preparing a zebrafish haploid cell line and a culture medium for preparing the zebrafish haploid cell line.

[0068] Method for preparing zebrafish haploid cell line

[0069] So far, no zebrafish haploid cell line has been successfully established in the art. This is mainly because the zebrafish haploid cell line is unstable and is prone to diploidization during the process of passage.

[0070] Therefore, in order to solve this problem, the present invention provides a method for preparing a zebrafish haploid cell line, comprising: preparing a zebrafish haploid cell line in the presence of a culture medium suitable for preparing a zebrafish haploid cell line. Preferably, the preparation method is a gynogenetic method.

[0071] The gynogenetic method mainly includes two steps: inactivation of sperm genetic material, and activation of eggs by sperm with inactivated genetic material to develop into haploid embryos.

[0072] Methods known in the art for inducing inactivation of sperm genetic material can be used, such as inducing inactivation of sperm genetic material by Coy rays, ultraviolet rays, and chemical drugs. Exemplary chemical drugs include, but are not limited to: toluidine blue, ethyleneurea, dimethylsulfate, trypalflavine, thiazine, etc.

[0073] Mature eggs have full developmental potential and can develop into embryos when fertilized with irradiated sperm.

[0074] Specifically, the method for preparing the zebrafish haploid cell line comprises:

[0075] 1) Inactivation of sperm genetic material;

[0076] 2) The egg is activated by the genetically inactivated sperm and develops into a haploid embryo;

[0077] 3) isolating and culturing cells at the blastocyst stage using a culture medium suitable for preparing a zebrafish haploid cell line; and

[0078] 4) Establish a stable zebrafish haploid cell line by subculturing.

[0079] In one or more embodiments, the zebrafish is a zebrafish of the ziwi:eGFP or kdrl:mcherry strain.

[0080] In one or more embodiments, in step 1), the inactivation of sperm genetic material is induced by Coy rays, ultraviolet rays, and chemicals (preferably, the chemicals include: toluidine blue, ethylene urea, dimethyl sulfate, acriflavine, thiazide). Preferably, the inactivation of sperm genetic material is induced by ultraviolet irradiation for 5 to 20 seconds (e.g., 5 to 15 seconds).

[0081] In one or more embodiments, in step 3), the blastocyst stage refers to the stage after 4 hours of development after fertilization, and the embryo has a multicellular morphology;

[0082] In one or more embodiments, in step 4), after passage to the 10th generation (e.g., the 25th generation), a stable zebrafish haploid cell line can be obtained. The stability of the zebrafish haploid cell line can be assessed using methods known in the art for evaluating genomic stability, such as, but not limited to, CNV analysis.

[0083] In one or more embodiments, the stage of embryo development is determined by observing two indicators, namely, the time of fertilization and the morphology. When the sperm and egg are added together and activated by adding egg water, fertilization begins, and the timer starts. When the development reaches 4 hours, it reaches the blastocyst stage. The embryo's morphology also gradually divides from the initial 1 cell to 2 cells, 4 cells, and finally to the multicellular blastocyst stage (such as Figure 4 The embryo in the figure has cells on the top and yolk on the bottom). The embryos have different shapes at different stages of development.

[0084] Medium suitable for the generation of zebrafish haploid cell lines

[0085] The present invention provides a culture medium suitable for preparing a zebrafish haploid cell line, which comprises a basal culture medium and an additive factor. In a preferred embodiment, the additive factor comprises: ZEE, basic FGF, Y-27632, BMP4. In a more preferred embodiment, the culture medium further comprises N2 supplement, B27 supplement, NEAA, 2-Mercaptoethanol, and L-glutamine. In a more preferred embodiment, the culture medium further comprises a serum substitute or serum, especially a serum substitute.

[0086] In one or more embodiments, the content (volume percentage) of the added factor is calculated based on the total volume of the culture medium:

[0087] ZEE: 1% to 10%; preferably 2% to 8%, 3% to 6% or 4% to 5% (e.g. 5%);

[0088] Basic FGF: 0.001% to 0.1%; preferably 0.001% to 0.05%, 0.01% to 0.05% or 0.005% to 0.2% (e.g. 0.01%);

[0089] Y-27632: 0.01% to 1%; preferably 0.01% to 0.5%, 0.05% to 0.3% or 0.05% to 0.2% (e.g. 0.1%);

[0090] BMP4: 0.001% to 0.1%; preferably 0.001% to 0.05%, 0.005% to 0.05% or 0.005% to 0.2% (eg 0.01%).

[0091] In one or more embodiments, the culture medium further comprises: N2 supplement, B27 supplement, NEAA, 2-Mercaptoethanol, and L-glutamine.

[0092] In one or more embodiments, the N2 supplement content is 0.1% to 5%; more preferably, 0.1% to 3%, 0.5% to 2% or 0.5% to 1.5% (eg, 1%).

[0093] In one or more embodiments, the content of B27 supplement is 0.1% to 5%; more preferably 0.1% to 3%, 0.5% to 2% or 0.5% to 1.5% (eg, 2%).

[0094] In one or more embodiments, the NEAA content is 0.1% to 5%; more preferably 0.1% to 3%, 0.5% to 2% or 0.5% to 1.5% (eg, 1%).

[0095] In one or more embodiments, the content of 2-Mercaptoethanol is 0.01% to 1%; more preferably 0.01% to 0.5%, 0.05% to 0.3% or 0.05% to 0.2% (eg, 0.1%).

[0096] In one or more embodiments, the L-glutamine content is 0.05-2.5 g / L; more preferably 0.05-1.5 g / L, 0.25-1 g / L, 0.25-0.75 g / L or 0.25-0.5 g / L (eg, 0.292 g / L).

[0097] In one or more embodiments, the culture medium further includes: a serum substitute or serum; preferably a serum substitute; preferably, its concentration is ≥5%, preferably ≥10%, and more preferably 5% to 20% (e.g., 5% to 15%, 10% to 20%, 15% to 20%, or 10% to 15%) based on the total volume of the culture medium. In one or more embodiments, the serum may be derived from an animal. The serum includes a serum extract, i.e., a substance extracted, purified or derived from the serum. In another one or more embodiments, the serum may also exist in the form of a serum substitute for industrial application. The serum substitute is a cell culture product that replaces serum; the components of the serum substitute include amino acids, metabolites, vitamins, trace elements, buffering capacity, recombinant growth factors, carbohydrates, etc., which are artificially prepared and have fixed components, but can replace animal-derived serum when used for culture.

[0098] In one or more embodiments, the culture medium also includes an antibiotic; preferably, it is an effective amount of penicillin and streptomycin, and more preferably a penicillin and streptomycin mixture containing glutamine (preferably, the effective amount of penicillin is 0.001% to 0.1%, more preferably 0.001% to 0.05%, 0.001% to 0.02% or 0.005% to 0.015%, for example 0.006%; wherein the effective amount of streptomycin is 0.001% to 0.1%, more preferably 0.001% to 0.05%, 0.001% to 0.02% or 0.005% to 0.015%, for example 0.01%).

[0099] In one or more embodiments, the basal culture medium includes a basal culture medium selected from the following group: DMEM, MEM, DMEM, DMEM / F12, Advanced DMEM / F12, RPMI; preferably, the culture medium includes a serum substitute, and the basal culture medium is a culture medium combined with a serum substitute (such as KO DMEM).

[0100] In a preferred embodiment of the present invention, the culture medium suitable for preparing the zebrafish haploid cell line is the culture medium shown in Table 2.

[0101] The present invention also provides the use of the additive factor in inhibiting the diploidization of haploid cells. In one or more embodiments, the inhibition is inhibiting the diploidization of haploid cells during the isolation, culture and / or passage process of haploid cells.

[0102] During the separation, culture and / or passage process of the haploid cells, the added factors provided by the present invention can inhibit the diploidization of haploid cells and maintain haploidy without affecting the normal growth of cells.

[0103] On the other hand, the present invention provides a composition for isolating, culturing and / or propagating (e.g., zebrafish) haploid cells, the composition comprising, but not limited to, a basic culture medium and ZEE, basic FGF, Y-27632, BMP4. In a more preferred embodiment, it also comprises N2 supplement, B27 supplement, NEAA, 2-Mercaptoethanol, L-glutamine. In a more preferred embodiment, it also comprises a serum substitute or serum, especially a serum substitute.

[0104] In one or more embodiments, based on the total volume of the composition, the content (volume percentage) of the added factor is:

[0105] ZEE: 1% to 10%; preferably 2% to 8%, 3% to 6% or 4% to 5% (e.g. 5%);

[0106] Basic FGF: 0.001% to 0.1%; preferably 0.001% to 0.05%, 0.01% to 0.05% or 0.005% to 0.2% (e.g. 0.01%);

[0107] Y-27632: 0.01% to 1%; preferably 0.01% to 0.5%, 0.05% to 0.3% or 0.05% to 0.2% (e.g. 0.1%);

[0108] BMP4: 0.001% to 0.1%; preferably 0.001% to 0.05%, 0.005% to 0.05% or 0.005% to 0.2% (eg 0.01%).

[0109] In one or more embodiments, the composition further comprises: N2 supplement, B27 supplement, NEAA, 2-Mercaptoethanol, L-glutamine;

[0110] Preferably, the N2 supplement content is 0.1% to 5%; more preferably 0.1% to 3%, 0.5% to 2% or 0.5% to 1.5% (e.g. 1%);

[0111] Preferably, the content of B27 supplement is 0.1% to 5%; more preferably, 0.1% to 3%, 0.5% to 2% or 0.5% to 1.5% (e.g., 2%);

[0112] Preferably, the NEAA content is 0.1% to 5%; more preferably 0.1% to 3%, 0.5% to 2% or 0.5% to 1.5% (e.g. 1%);

[0113] Preferably, the content of 2-Mercaptoethanol is 0.01% to 1%; more preferably, 0.01% to 0.5%, 0.05% to 0.3% or 0.05% to 0.2% (e.g., 0.1%);

[0114] Preferably, the L-glutamine content is 0.05-2.5 g / L; more preferably, 0.05-1.5 g / L, 0.25-1 g / L, 0.25-0.75 g / L or 0.25-0.5 g / L (for example, 0.292 g / L).

[0115] In one or more embodiments, the composition further includes: a serum substitute or serum; preferably a serum substitute; preferably, the concentration thereof is ≥5%, preferably ≥10%, and more preferably 5% to 20% (e.g., 5% to 15%, 10% to 20%, 15% to 20%, or 10% to 15%) based on the total volume of the culture medium.

[0116] In one or more embodiments, the composition also includes an antibiotic; preferably, it is an effective amount of penicillin and streptomycin, and more preferably a penicillin and streptomycin mixture containing glutamine (preferably, the effective amount of penicillin is 0.001% to 0.1%, more preferably 0.001% to 0.05%, 0.001% to 0.02% or 0.005% to 0.015%, for example 0.006%; wherein the effective amount of streptomycin is 0.001% to 0.1%, more preferably 0.001% to 0.05%, 0.001% to 0.02% or 0.005% to 0.015%, for example 0.01%).

[0117] The present invention also provides the use of the composition for preparing, isolating, culturing and / or passage of zebrafish haploid cell lines.

[0118] The present invention also provides a kit, which comprises the composition of the present invention, or the culture medium of the present invention suitable for preparing the zebrafish haploid cell line.

[0119] Zebrafish haploid cell lines and their applications

[0120] The present invention provides a zebrafish haploid cell line, which is prepared by the preparation method described in any technical solution of the present invention.

[0121] In another aspect, the present invention also provides the use of zebrafish haploid cells obtained by the preparation method described in any technical solution of the present invention in genetic research (e.g., genetic screening, gene knockout / knock-in, preparation of semi-cloned zebrafish). Due to the characteristics of the haploid single-copy genotype, the preparation method of the present invention and the obtained haploid cells provide a good and efficient genetic model for studying stem cells, embryonic development, cell signaling pathways, etc.

[0122] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. 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.

[0123] Unless otherwise specified, all percentages, ratios or proportions in the content are by volume. The units of volume percentage in the present invention are well known to those skilled in the art, for example, it refers to the volume (ml) of solute in 100 ml of solution.

[0124] Abbreviation

[0125] PG72ziwi: a zebrafish haploid cell line

[0126] ZF4: zebrafish embryonic fibroblast cell line (diploid cell line).

[0127] Experimental Materials

[0128] The zebrafish used in the examples, their sources, and culture medium components are shown in Table 1.

[0129] Table 1

[0130]

[0131] Other experimental materials used in the examples:

[0132] TryPLE; 1mg / ml pronase; Matrigel; BSA; PBS, DMSO; colchicine; bleach; RNAase; PI dye; MS-222 anesthesia; 75% alcohol; 10cm dish, centrifuge tube, pipette, dropper, timer, cryotube.

[0133] Egg water: Add 1.5 ml of stock salts to 1 L of distilled water.

[0134] Stock Salts: Prepare by adding 40g sea salt to 1L distilled water.

[0135] Experimental Instruments

[0136] Clean bench, centrifuge, dissecting microscope, ZF-5 portable UV lamp (wavelength 254nm), carbon dioxide incubator, flow cytometer, UV irradiation device, refrigerator.

[0137] Experimental methods

[0138] 1. Preparation of embryo extract (ZEE)

[0139] 1.1 Culture AB strain zebrafish embryos for 72 hours and collect 400 embryos into a 15 ml centrifuge tube;

[0140] 1.2 Wash the embryos 5 times with PBS;

[0141] 1.3 Transfer the embryos to a 1.5 ml centrifuge tube, add 500 μl KO DMEM, and grind evenly using an electric grinder;

[0142] 1.4 Centrifuge at low temperature and high speed to separate the grinding solution into layers, take the transparent extract solution in the middle, and adjust the volume to 1 ml. The final concentration of the embryo extract is 400 embryos / ml;

[0143] 1.5 Sterilize by filtering with a 0.22 μm filter and store at -80°C.

[0144] 2. Preparation of Matrigel-coated plates

[0145] According to the manufacturer's standard of Matrigel coated plate (purchased from Corning-Biocoat, catalog number 354277), dilute with KODMEM, add the diluted solution to the culture plate, place in a 28°C incubator for coating for at least 30 minutes, discard the coating solution when using, and wash with PBS.

[0146] 3. Method for establishing zebrafish haploid embryonic cell line

[0147] 3.1 Preparation of haploid zebrafish embryos

[0148] 3.1.1 Take 4 adult ziwi-GFP male zebrafish and anesthetize them with MS-222.

[0149] 3.1.2 Use absorbent paper to absorb the water from the zebrafish and place it in the paper slot.

[0150] 3.1.3 Use a 20 μl white pipette tip to absorb a small amount of PBS and moisten the reproductive opening of the zebrafish.

[0151] 3.1.4 Gently pinch with your fingers and slowly suck out the liquid. A small amount of white liquid can be seen on the tip of the gun.

[0152] 3.1.5. Transfer the sperm into 300 μl of PBS.

[0153] 3.1.6 Take 300 μl of sperm and add it to the center of the concave lens, then place the concave lens on a dish filled with ice.

[0154] 3.1.7 Irradiate with UV light for 10 seconds.

[0155] 3.1.8 After irradiation, transfer the samples to new centrifuge tubes and store them on ice.

[0156] 3.1.9 Take an adult female zebrafish, anesthetize it with MS-222, and dry the fish with absorbent paper.

[0157] 3.1.10 Place the eggs in a clean and dry culture dish and press lightly on the fish abdomen with the tip of your ring finger to push the eggs out from the middle toward the reproductive opening.

[0158] 3.1.11 Place about 100 eggs in each culture dish, pipette 50ul of sperm preparation solution and drop it on the eggs to completely soak them.

[0159] 3.1.12 Take 1 ml of water (to activate sperm and combine with egg), drop it on the egg and shake the culture dish for 10 seconds.

[0160] 3.1.13 Transfer the culture dish to a 28.5℃ incubator for culture.

[0161] 3.2 Preparation of CM_BMP4 cell culture medium

[0162] Prepare CM_BMP4 cell culture medium according to Table 2, and prepare CM cell culture medium according to Table 3. In the subsequent steps, CM_BMP4 cell culture medium was used unless otherwise specified.

[0163] Table 2. CMBMP4 cell culture medium

[0164]

[0165]

[0166] Table 3. CM cell culture medium

[0167]

[0168] Note: The 100X Penicillin / streptomycin+L-glutamine (Penicillin-Streptomycin-Glutamine) solution in Tables 2 and 3 contains 10,000 units of penicillin, 10,000 μg of streptomycin, and 29.2 mg / mL of L-glutamine, dissolved in 10 mM citrate buffer (to maintain a stable pH value), 50 mg / mL. After dilution at 1:100, the final concentration is 0.5 g / L (including 0.292 g / L of L-glutamine).

[0169] 3.3 Isolation of haploid embryonic cells

[0170] 3.3.1 Preparation of haploid embryos by artificial insemination

[0171] 3.3.2 When isolating 3.3hpf embryonic cells, wait until the fertilized eggs develop to 2.5hpf, and collect embryos that develop normally and at the same stage in 2ml EP tubes, with about 200-300 embryos in each EP tube. If isolating embryonic cells at other time points, collect normal developing embryos in advance accordingly.

[0172] 3.3.3 The collected embryos were transferred to the cell room for disinfection and sterilization. They were washed twice with sterile PBS, disinfected with 0.1% bleach for 2 minutes, and then washed twice with PBS. If embryos at other time points were to be isolated, they were treated with 75% alcohol for 15 seconds before bleach disinfection, and then washed several times with PBS.

[0173] 3.3.4 Add 1 ml of preheated 1 mg / ml pronase to the EP tube and incubate at 37°C for 5 min.

[0174] 3.3.5 Aspirate the chorion and enzyme solution, gently blow the embryo with sterile PBS to fully separate the chorion, wash twice with PBS, and transfer to a 60mm culture dish containing PBS.

[0175] 3.3.6 Use forceps and a 1ml syringe to quickly peel off the cell clusters, collect the cells, gently blow the cells to mix, add CM_BMP4 medium, and plate them for culture. There are about 15 embryonic cells in a 6-well plate and about 2-4 embryonic cells in a 96-well plate. The cell culture dish needs to be coated with Matrigel 30 minutes in advance.

[0176] 3.3.7 After plating, detailed information on cell treatment should be recorded, including date, fish strain, embryo ploidy, separation time, substrate, cell culture medium, number of embryos per well and cell density, cell culture plate specifications, and observation date.

[0177] 3.4 Cell passaging

[0178] 3.4.1 The first generation of cell lines takes a long time to culture, which may take 8-10 days for the primary cells to become stable. Passaging too early will cause the cell line to be lost. Use the pancreatic enzyme substitute TryPLE to digest the cells fully into single cells. After they are separated from the bottom of the culture dish, gently blow them. No neutralization is required. Centrifuge at 200g for 3 minutes to collect the cells. Resuspend the cells in CM_BMP4 medium, and the passaging ratio is 1:2.

[0179] 3.4.2 Subsequent subculture process The cells are subcultured according to their growth status and cell density. Generally, the subculture density should not exceed 1:5. The growth density of the cells after subculture should be able to proliferate to 90% confluence within about 5 days. The zebrafish haploid cell line grows slowly during the first few generations of culture, and the subculture time is correspondingly longer.

[0180] 3.5 Cryopreservation and thawing of haploid cells

[0181] 3.5.1 Cell cryopreservation

[0182] When the cells are passaged to a 6-well plate or a 6 cm culture dish and grown to about 90% confluence, the cells are frozen. Before freezing, the haploid cell line needs to be analyzed for haploid ratio by FACS. Cells with too low a ratio need to be sorted and grown to a sufficient number before freezing. Otherwise, the damage to the cells by freezing may lead to a further reduction in haploid cells, thereby losing the haploid cell line. The specific method is as follows: Digest the cells with the pancreatic enzyme substitute TryPLE. After the cells are digested into single cells, gently blow and centrifuge at 200g for 3 minutes to collect the cells. Resuspend the cells in freezing solution (90% CM_BMP4 medium + 10% DMSO) and transfer them to a cryopreservation tube. Record the cell line name, ploidy, strain, date, passage number and freezing density on the cryopreservation tube, and a transparent tape can be attached to prevent the loss of the label. Finally, put the cells into a freezing box and transfer them to a -80°C refrigerator and liquid nitrogen for storage.

[0183] 3.5.2 Cell recovery

[0184] Before cell recovery, the culture dish needs to be coated with Marigel solution at 28.5℃ for more than 30 minutes. After taking the cells out of the -80℃ freezer or liquid nitrogen, quickly place them in a 37℃ water bath and shake them back and forth to thaw the cells quickly. Add CM_BMP4 medium to wash the cells to remove DMSO in the cryopreservation solution. Collect the cells by centrifugation at 200g for 3 minutes, resuspend the cells in CM_BMP4 medium, and plate them according to the needs and cell density.

[0185] 3.6 Identification of haploid cells

[0186] 3.6.1 Karyotype analysis

[0187] 3.6.1.1 Observe the cell status under a microscope. On the third day of culture and subculture, when the confluence is about 70%, add colchicine at a final concentration of 0.4μg / ml for 8h. If the colchicine treatment time is insufficient, the cells will be in the M mid-phase, and it will be difficult to find a suitable karyotype analysis chart after dropping. If the treatment time is too long, the cells will die and become suspended.

[0188] 3.6.1.2 After collecting the cells, centrifuge at 200g for 3 minutes to remove the supernatant, add 1ml of hypotonic solution to resuspend the cells, and then add 4ml of hypotonic solution to mix thoroughly. The concentration of the hypotonic solution is 0.075M KCl (0.56%), which is placed in a 28.5℃ water bath to preheat. During the hypotonic process, use a pipette to gently suspend the precipitated cells from time to time. Insufficient hypotonic time will cause severe cytoplasmic staining when the cells are stained, and the chromosomes will be difficult to spread and have a smaller shape.

[0189] 3.6.1.3 After the hypotonic treatment is completed, add 1 ml of freshly prepared 4℃ precooled fixative to 4 ml of hypotonic cell suspension. The fixative is a 3:1 ratio of methanol to glacial acetic acid. Glacial acetic acid has strong penetration and rapid fixation, but it easily causes tissue expansion, while formaldehyde can cause tissue contraction. The combination of the two has the best effect. Gently blow with a dropper to mix well and place for 5 minutes, then centrifuge at 1000g for 10 minutes.

[0190] 3.6.1.4 Discard the supernatant, add 1 ml of fresh pre-cooled fixative, blow gently to mix into a single cell suspension, then add enough fixative according to the cell volume (add 5 ml of fixative for 6 cm), fix at 4°C for more than 30 minutes, then centrifuge at 1000g for 10 minutes, repeat twice.

[0191] 3.6.1.5 Discard the supernatant, add a small amount of 1 ml of fixative, and make a drop sample slide for observation.

[0192] 3.6.2 Ploidy analysis

[0193] 3.6.2.1 Digest the cells cultured in 6-well or 12-well plates with TryPLE, optionally add 3% BSA for neutralization, and centrifuge at 200g for 3 minutes to collect the cell pellet.

[0194] 3.6.2.2 Resuspend the cells with 300μl pre-cooled PBS, and mix them by repeated pipetting to form single cells. Set the vortex mixer to low setting, vortex mix the cells, slowly add 700μl of anhydrous ethanol to a final concentration of 70%, and place at 4℃ to fix overnight. It can be stored at -20℃ for a month.

[0195] 3.6.2.3 The fixed cells were centrifuged at 2500rpm for 5min in a horizontal centrifuge, and washed twice with 500ul pre-cooled PBS at 2000rpm for 2min. After washing, resuspended with 200ul 20ug / ml RNAase PBS, and digested in a 37℃ water bath for 30min. Every 10min, tap the bottom of the EP tube to prevent the cells from sinking. (Take 3ul 33mg / ml Sigma RNAase stock solution, dilute it with 4.997ml PBS to 20ug / ml, and dispense it into 25x200ul / tube, stored at -20℃).

[0196] 3.6.2.4 Add 200ul 50ug / ml PI buffer, wrap in foil and stain for 10min at 4℃ away from light. Try to test immediately after staining to avoid agglomeration. If there are many samples, add PI staining solution in batches before testing. (It takes about 60min to remove fixed cells and test them. 2mg / ml PI staining solution, dilute with PBS to 100ug / ml 2x working solution, 20× dilution, take 100ul PI + 1900ul PBS, store at 4℃ away from light).

[0197] 3.6.2.5 After thoroughly mixing the cells, filter them with a 200-mesh filter and use Beckman CytoFlex for FACS detection.

[0198] 3.6.2.6 Detect red fluorescence at an excitation wavelength of 488nm and detect light scattering at the same time. Adjust the acquisition parameter settings and save as a template. Adjust FSC and B610-ECD to the appropriate position. Adjust to 1n\2n\4n in the appropriate range and record 10,000 cells at a medium flow rate. When exporting and saving files, you need to export the xit file and the data folder containing fcs at the same time. Use CytExpert2.3 to analyze DNA content.

[0199] 3.6.3 Genome stability (CNV) analysis

[0200] Genomic DNA was extracted from haploid cell samples using QIAamp DNA Mini. The extracted DNA was quantified and integrity checked. gDNA was randomly sheared using an ultrasonic disruptor (Diagenode Belgium) and purified using VAHTSDNAClean Beads. DNA was repaired and end-adapted using multi-step enzymatic reactions, and the purified Adapterligation products were amplified for raw library. The library was sequenced on the Illumina Novaseq6000 platform, and the raw data were processed using fastp and fastqc for quality control. Clean reads were aligned to the zebrafish genome using Sentieon bmem, sorted, and repetitive sequences were removed. Single nucleotide variants (SNVs) and indexing were analyzed using Sentieon DNAscope, copy number variations were detected using Control-freeC (v11.6), and structural variations were identified using Lumpy (v0.2.13).

[0201] Example 1. Preparation of zebrafish haploid cell line

[0202] In this embodiment, a method for preparing a zebrafish haploid cell line is provided, comprising:

[0203] 1) Genetic inactivation of sperm chromosomes through ultraviolet (UV) irradiation;

[0204] 2) The egg is activated by the genetically inactivated sperm and develops into a haploid embryo;

[0205] 3) isolating and culturing cells at the blastocyst stage using a culture medium suitable for preparing a zebrafish haploid cell line; and

[0206] 4) Establish stable cell lines through subculturing.

[0207] The flow chart of the method for preparing zebrafish haploid cell lines is as follows Figure 2 The specific preparation method is shown in Part 3 of the Experimental Methods.

[0208] Step 2) 24 hours after the eggs were fertilized and developed into haploid embryos, the morphology of the zebrafish embryos was observed and the results were compared with those of diploid embryos that had not been irradiated with UV. Figure 3 As shown, 24 hours after zebrafish embryo fertilization, haploid embryos were slow to develop and their body length was significantly shorter than that of diploid embryos, which also indicates that UV irradiation inactivated sperm DNA.

[0209] Next, the morphology of zebrafish blastocyst embryos was observed, and step 3) was performed to isolate blastocyst cells and observe the morphology of cells at 0 h, 6 h, and 24 h of culture. Figure 4 As shown in A. The cells were then passaged to observe the cell morphology of the 60th generation zebrafish haploid cell line, and the ploidy of the 57th generation zebrafish haploid cells was analyzed by flow cytometry. The results showed that at 0h, the cells just separated were dispersed single cells, and after 6h of culture, several smaller cell clusters were formed, and after 24h of culture, larger cell clusters were formed. They all grew adherently, and it can be seen that the cells are constantly dividing and growing.

[0210] Example 2: Cell morphology and identification results of zebrafish haploid cell lines

[0211] After obtaining the zebrafish haploid cell line using the preparation method of Example 1, the inventors named it PG72ziwi (PG is the abbreviation of parthenogenetic haploid, and ziwi represents the zebrafish strain) and observed the cell morphology of the obtained zebrafish haploid cell line. Figure 1 As shown, compared with the haploid cells of medaka ( Figure 1 A), Figure 1 The cells in A are round or polygonal in shape, with little cytoplasm and a large nucleus, and the nucleolus can be clearly seen; Figure 1 Although the cell in B is also polygonal in shape, the nucleus is not obvious, and the nucleolus is not visible, and there is more cytoplasm, which shows that the zebrafish haploid cell line obtained in the present invention is completely different from it.

[0212] Next, the inventors identified the obtained zebrafish haploid cell line. The results of karyotype analysis of haploid cells showed that zebrafish haploid cells contained 25 chromosomes. The results of karyotype analysis of the 28th generation zebrafish haploid cells showed that cells with chromosome numbers between 22-29 accounted for more than 80%, while cells with chromosome numbers between 48-54 accounted for only a dozen percent, indicating that most cells are haploid and only a small number of cells are diploid. The 25th generation (P25) and 57th generation (P47) zebrafish haploid cell lines were compared with the commercial ZF4 diploid cell line by genome stability (CNV) analysis, including karyotype analysis (chromosome number determination method), ploidy analysis (DNA content) or transcriptome level.

[0213] From the morphological point of view, ZF4 is a fibroblast-like cell, while the cell morphology of PG72ziwi is similar to that of epithelial cells. From the results of genome stability (CNV) analysis, the genome of the zebrafish haploid cell line established by the present invention is relatively stable, and there is little difference between the 25th generation (P25) and 57th generation (P47) zebrafish haploid cell lines, with only slight changes in chromosomes 4, 6, and 19. The top 10 genes with the highest expression levels in the zebrafish haploid embryonic cell line were analyzed. Among the top 10 genes with the highest expression levels, 4 are related to mitochondrial energy metabolism, among which the cytochrome c oxidase I (mt-co1) gene has the highest expression level ( Figure 6 ). In addition, there are genes related to cytoskeleton, ribosome complex, and lectin.

[0214] Example 3: Optimization of culture medium for preparing zebrafish haploid cell line

[0215] So far, no zebrafish haploid cell line has been successfully established, partly because haploid cell lines are very prone to diploidization. Therefore, in this example, the effect of the culture medium for preparing the zebrafish haploid cell line on the maintenance of the ploidy of the zebrafish haploid cells was investigated.

[0216] According to Table 2 and Table 3, a cell culture medium (CM_BMP4) supplemented with BMP4 and a cell culture medium (CM) without BMP4 were prepared respectively. In step 3), CM_BMP4 and CM culture medium were used to separate and culture cells, and the percentage of G1 cells in the zebrafish haploid cells during the subculture process in step 4) was observed. The results are shown in FIG. Figure 8 As shown in A. CM_BMP4 medium has a higher proportion of haploid cells in the G1 phase compared to CM medium, indicating that BMP4 can promote the maintenance of haploidy.

[0217] The inventor's experiments show that each component in the culture medium plays an overall promoting role. Based on the components in Table 2, if NEAA is removed, cell growth will be restricted, because NEAA is a non-essential amino acid and a raw material for protein synthesis. If NEAA is lacking, cells will not be able to synthesize the proteins they need and will not be able to divide. If ZEE is removed while the other components in Table 2 remain unchanged, the cell proliferation activity is poor and almost no growth occurs ( Figure 7 ). If Y-27632 is removed, primary cells will not be able to aggregate to form cell clusters, and cell lines cannot be established. Therefore, each component in the culture medium plays an integral role and cannot be removed.

[0218] Example 4: Effect of zebrafish strain on preparation of zebrafish haploid cell line

[0219] In this example, the effect of zebrafish strain on the preparation of zebrafish haploid cell lines was investigated. By comparing four different zebrafish strains, the AB strain, and the transgenic (Tg) ziwi:eGFP, sox17:eGFP, and kdrl:mcherry strains, it was found that although haploid cell lines of the AB and sox17:eGFP transgenic strains could be established, they were relatively prone to diploidization; while the ziwi:eGFP and kdrl:mcherry strains were significantly more stable ( Figure 8 B. Figure 8 C).

[0220] Example 5: Application of zebrafish haploid cell line

[0221] The zebrafish haploid cell line of the present invention is not prone to diploidization, is relatively stable, and can be applied to genetic screening, gene knockout / knock-in, transplantation cloning, and the like.

[0222] 1. Genetic screening

[0223] The zebrafish haploid cell line of the present invention can be used for genetic screening of toxins, signal pathways, and small molecule compounds. First, a cell mutation library is established, and then drug treatment is performed. The surviving cell clones are sequenced and analyzed to find the mutant genes, and knockout verification is performed to find genes that play important functions in toxins or signal pathways.

[0224] 2. Gene knockout / knock-in

[0225] The zebrafish haploid cell line of the present invention is used to knock out genes or knock in genes by homologous recombination or CRISPR-Cas9 method. Specifically, the inventors inserted the red fluorescent protein gene mcherry into the 3' end of the krt94 gene and successfully expressed red fluorescence.

[0226] 3. Preparation of semi-cloned fish

[0227] The zebrafish haploid cell line of the present invention is used to prepare semi-cloned fish. Specifically, the inventors transplanted haploid cell nuclei into unfertilized fish eggs, fertilized the eggs and developed them into semi-cloned fish, thereby obtaining semi-cloned fish carrying the donor genome.

[0228] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims. At the same time, all the documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference separately.

Claims

1. A method for preparing a zebrafish haploid cell line, comprising: 1) Inactivation of sperm genetic material; 2) The egg is activated by the genetically inactivated sperm and develops into a haploid embryo; 3) isolating and culturing cells at the blastocyst stage using a culture medium suitable for preparing a zebrafish haploid cell line; and 4) Establish a stable zebrafish haploid cell line by subculturing.

2. The preparation method according to claim 1, characterized in that The culture medium suitable for preparing the zebrafish haploid cell line comprises a basic culture medium and added factors: ZEE, basic FGF, Y-27632, and BMP4.

3. The preparation method according to claim 2, characterized in that: Calculated based on the total volume of the culture medium, the content of the added factor is: ZEE: 1% to 10%; preferably 2% to 8%, 3% to 6% or 4% to 5%; Basic FGF: 0.001% to 0.1%; preferably 0.001% to 0.05%, 0.01% to 0.05% or 0.005% to 0.2%; Y-27632: 0.01% to 1%; preferably 0.01% to 0.5%, 0.05% to 0.3% or 0.05% to 0.2%; BMP4: 0.001% to 0.1%; preferably 0.001% to 0.05%, 0.005% to 0.05% or 0.005% to 0.2%.

4. The preparation method according to claim 2 or 3, characterized in that: The culture medium further comprises: N2 supplement, B27 supplement, NEAA, 2-Mercaptoethanol, L-glutamine; Preferably, the N2 supplement content is 0.1% to 5%; more preferably 0.1% to 3%, 0.5% to 2% or 0.5% to 1.5%; Preferably, the content of B27 supplement is 0.1% to 5%; more preferably, 0.1% to 3%, 0.5% to 2% or 0.5% to 1.5%; Preferably, the NEAA content is 0.1% to 5%; more preferably, 0.1% to 3%, 0.5% to 2% or 0.5% to 1.5%; Preferably, the content of 2-Mercaptoethanol is 0.01% to 1%; more preferably, 0.01% to 0.5%, 0.05% to 0.3% or 0.05% to 0.2%; Preferably, the L-glutamine content is 0.05 to 2.5 g / L; more preferably, 0.05 to 1.5 g / L, 0.25 to 1 g / L, 0.25 to 0.75 g / L or 0.25 to 0.5 g / L.

5. The preparation method according to any one of claims 2 to 4, characterized in that: The culture medium further comprises: a serum substitute or serum; preferably a serum substitute; preferably, the concentration thereof is ≥5%, preferably ≥10%, more preferably 5% to 20% based on the total volume of the culture medium; or The culture medium also includes antibiotics; preferably, it is an effective amount of penicillin and streptomycin, more preferably a penicillin and streptomycin mixture containing glutamine; preferably, the effective amount of penicillin is 0.001% to 0.1%, more preferably 0.001% to 0.05%, 0.001% to 0.02% or 0.005% to 0.015%; wherein the effective amount of streptomycin is 0.001% to 0.1%, more preferably 0.001% to 0.05%, 0.001% to 0.02% or 0.005% to 0.015%.

6. The preparation method according to any one of claims 2 to 5, characterized in that: The basal culture medium includes a basal culture medium selected from the following group: DMEM, MEM, DMEM / F12, Advanced DMEM / F12, and RPMI; preferably, the culture medium includes a serum substitute, and the basal culture medium is a culture medium combined with a serum substitute.

7. The method for preparing a zebrafish haploid cell line according to claim 1, characterized in that: The zebrafish is a zebrafish of the ziwi:eGFP or kdrl:mcherry strain.

8. The method for preparing a zebrafish haploid cell line according to claim 1, characterized in that: The preparation method comprises one or more of the following AC: A. In step 1), the inactivation of sperm genetic material is induced by Coy rays, ultraviolet rays, or chemical drugs. Preferably, the inactivation of sperm genetic material is induced by ultraviolet irradiation for 5 to 20 seconds; B. In step 3), the blastocyst stage refers to the stage 4 hours after fertilization, when the embryo is multicellular in morphology; C. In step 4), a stable zebrafish haploid cell line was obtained after 10 generations.

9. A zebrafish haploid cell line, prepared by the method according to any one of claims 1 to 8.

10. A composition suitable for preparing, isolating, culturing and / or propagating a zebrafish haploid cell line, comprising: Basic culture medium and added factors: ZEE, basic FGF, Y-27632, BMP4; Preferably, the content of the added factor is calculated based on the total volume of the composition: ZEE: 1% to 10%; preferably 2% to 8%, 3% to 6% or 4% to 5%; Basic FGF: 0.001% to 0.1%; preferably 0.001% to 0.05%, 0.01% to 0.05% or 0.005% to 0.2%; Y-27632: 0.01% to 1%; preferably 0.01% to 0.5%, 0.05% to 0.3% or 0.05% to 0.2%; BMP4: 0.001% to 0.1%; preferably 0.001% to 0.05%, 0.005% to 0.05% or 0.005% to 0.2%.

11. The composition according to claim 10, characterized in that The composition further comprises: N2 supplement, B27 supplement, NEAA, 2-Mercaptoethanol, L-glutamine; Preferably, the N2 supplement content is 0.1% to 5%; more preferably 0.1% to 3%, 0.5% to 2% or 0.5% to 1.5%; Preferably, the content of B27 supplement is 0.1% to 5%; more preferably, 0.1% to 3%, 0.5% to 2% or 0.5% to 1.5%; Preferably, the NEAA content is 0.1% to 5%; more preferably, 0.1% to 3%, 0.5% to 2% or 0.5% to 1.5%; Preferably, the content of 2-Mercaptoethanol is 0.01% to 1%; more preferably, 0.01% to 0.5%, 0.05% to 0.3% or 0.05% to 0.2%; Preferably, the L-glutamine content is 0.05 to 2.5 g / L; more preferably, 0.05 to 1.5 g / L, 0.25 to 1 g / L, 0.25 to 0.75 g / L or 0.25 to 0.5 g / L.

12. The composition according to claim 10 or 11, characterized in that The composition further comprises: a serum substitute or serum; preferably a serum substitute; preferably, the concentration thereof is ≥5%, preferably ≥10%, more preferably 5% to 20% based on the total volume of the culture medium; or The composition also includes an antibiotic; preferably, it is an effective amount of penicillin and streptomycin, more preferably a penicillin and streptomycin mixture containing glutamine; preferably, the effective amount of penicillin is 0.001% to 0.1%, more preferably 0.001% to 0.05%, 0.001% to 0.02% or 0.005% to 0.015%; wherein the effective amount of streptomycin is 0.001% to 0.1%, more preferably 0.001% to 0.05%, 0.001% to 0.02% or 0.005% to 0.015%.

13. Use of a composition for preparing, isolating, culturing and / or propagating a zebrafish haploid cell line; wherein the composition is the composition according to any one of claims 10 to 12.

14. A kit comprising the composition according to any one of claims 10 to 12, or comprising a culture medium suitable for preparing a zebrafish haploid cell line as defined in any one of claims 1 to 8.

15. Use of the zebrafish haploid cell line according to claim 9 for genetic screening, gene knockout / knock-in, and preparation of semi-cloned fish.

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