Method for preparing all-male offspring chicken

Abstract

The invention discloses a method for preparing an all-male offspring chicken, which comprises the following steps: separating primordial germ cells from a male chicken embryo gonad, carrying out single embryo line establishment, and injecting the cells into an HSV gene edited chicken of which endogenous primordial germ cells are eliminated by ganciclovir induction to prepare a chimeric cock and a chimeric hen. And finally, copulating the chimeric cocks and the chimeric hens to obtain all-male progeny chickens. According to the invention, the problem of uneven quality of chicks in the traditional chicken breeding industry is solved, and a feasible scheme is provided for exclusively copying excellent male individuals. The chicks produced by the method are uniform in genetic background, can stably inherit excellent characters of excellent male parents, and are of great significance to protection and propagation of excellent cock resources.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a method for preparing all-male offspring chickens. Background Art

[0002] With the rapid development of the chicken breeding industry, the market demand for high-quality chickens and their products is increasing day by day. At present, a flock of roosters with excellent and uniform traits is still an important requirement in the modern chicken breeding industry. Different from mammals, due to their special reproductive physiological characteristics, such as the fertilized egg developing to the late blastocyst stage when discharged and being closely connected to the yolk to absorb nutrients, it is impossible for poultry to clone individuals with exactly the same genotype through somatic cell nuclear transfer technology like mammals. However, poultry can transfer genetic information to the next generation through primordial germ cell (PGC) transplantation. Therefore, the excellent genetic characteristics of roosters can be passed on through PGC transplantation.

[0003] The sex determination of both poultry and mammals is controlled by genetic factors, and the sex-determining chromosomes are sex chromosomes. The difference is that in mammals, the female has homozygous X / X sex chromosomes and produces homozygous gametes X eggs, while the male has heterozygous X / Y sex chromosomes and produces heterozygous gametes X and Y sperm; in poultry, on the contrary, the female has heterozygous Z / W sex chromosomes and produces heterozygous gametes Z and W eggs, and the male has homozygous Z / Z sex chromosomes and produces homozygous gametes Z sperm. Research shows that animal PGCs have varying degrees of bisexual developmental potential when differentiating into gametes. The developmental direction of gametes does not completely depend on the sex chromosome composition of the germ cells themselves, but can be regulated by the gonadal environmental factors to develop into sperm or eggs. In poultry, when male chicken PGCs (Z / Z) are transplanted into female (Z / W) recipient embryos, the male-derived germ cells can successfully complete oogenesis and obtain functional Z oocytes under the support of the ovarian environment.

[0004] The HSV gene-edited homozygous roosters used in this study were prepared by using the CRISPR / cas9-mediated gene site-directed knock-in method to target and knock in the herpes simplex virus thymidine kinase gene (HSV-TK) at the 3' end of the DAZL gene specifically expressed in germ cells, and a EGFP fluorescent reporter gene was inserted to observe the PGCs after gene modification, and HSV-TK gene-edited chickens that can specifically eliminate endogenous germ cells were successfully prepared. Since HSV-TK phosphorylates non-toxic ganciclovir (GCV), and then the cellular kinase phosphorylates GCV again to produce GCV triphosphate, which inhibits DNA elongation and cell division and induces apoptosis, ultimately leading to cell death. Therefore, when ganciclovir was added to HSV-TK-GFP chicken embryos, it was found that the green fluorescent germ cells were specifically eliminated in both female and male individuals during the embryonic period and after hatching, but the development of gonadal somatic cells was not affected.

[0005] Therefore, through the establishment of a single-embryo PGC cell line and PGC transplantation technology, the present invention obtains chimeric chickens carrying PGCs of the same male chicken embryo, pairs the roosters and hens of the chimeras, and finally obtains all-male offspring chickens with relatively homogeneous genetic backgrounds. Compared with traditional inbreeding, the single-parent offspring are expected to retain or restore most of the genetic information of a specific donor rooster. This production technology can solve the problem of uneven quality of chicken seedlings in production, and also provides a feasible solution for specifically replicating excellent male individuals, offering new possibilities for the expansion and optimization of breeding chicken resources. Summary of the Invention

[0006] The object of the present invention is to overcome the deficiencies in the prior art and provide a method for preparing all-male offspring chickens, which can solve the problem of uneven quality of chicken seedlings in production and provide a feasible solution for specifically replicating excellent male individuals.

[0007] To achieve the above object, the technical solution provided by the present invention is as follows:

[0008] Step S1, cell collection: Isolate male primordial germ cells from the gonads of a single 7-day-old hatched chicken embryo to establish a male primordial germ cell line.

[0009] Step S2, preparation of chimeric roosters and chimeric hens: Inject male primordial germ cells into HSV gene-edited chicken embryos that have been induced to eliminate endogenous primordial germ cells with ganciclovir to obtain chimeric roosters and chimeric hens.

[0010] Step S3, production of all-male offspring: Mate chimeric roosters with chimeric hens to obtain all-male offspring chickens.

[0011] Further, the method for cell collection in step S1 is as follows: Take out the gonads from a single male chicken embryo, digest the gonads with trypsin, add mKO culture medium, centrifuge and take the supernatant and place it in an incubator for adherent treatment for 6 hours. After the adherent treatment is completed, centrifuge again and take the supernatant for the proliferation and passage of primordial germ cells to establish a male primordial germ cell line.

[0012] Further, step S2 includes the following steps:

[0013] Step S21, collect semen from HSV gene-edited homozygous roosters and breed them with wild-type hens, and collect HSV eggs.

[0014] Step S22, place the collected eggs in a constant-temperature incubator for 2 days, make a small opening in the air chamber of the eggs, inject the male primordial germ cells obtained in step S1 into the heart of the gene-edited chicken embryos with a syringe, and at the same time inject ganciclovir into the chicken embryos to specifically eliminate endogenous germ cells; among them, 10,000 male primordial germ cells are injected into each egg.

[0015] Step S23, seal the notch of the hatching egg with a sealing film, and put the hatching egg back into the incubator until the chick hatches, obtaining chimeric roosters and chimeric hens.

[0016] Among them, the preparation method of the HSV gene-edited rooster is as follows: using the CRISPR / cas9-mediated gene site-directed knock-in technology, the herpes simplex virus thymidine kinase gene (HSV-TK) is targeted to knock into the 3' end of the DAZL gene specifically expressed in germ cells, and an EGFP fluorescent reporter gene is connected to observe the primordial germ cells after gene modification, and then the HSV-TK gene-edited chicken that can specifically eliminate endogenous germ cells can be successfully prepared.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention provides a method for preparing all-male offspring chickens. The chicken seedlings produced by this method are uniform, can effectively inherit the excellent traits of the excellent male parent, and the all-male offspring are beneficial to the protection and expansion of excellent rooster resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the male primordial cell germ line;

[0020] Figure 2 is the identification result of the male primordial cell germ line;

[0021] Figure 3 is to eliminate the endogenous primordial cells of the HSV gene-edited recipient chicken embryo. DETAILED DESCRIPTION OF THE INVENTION

[0022] The following is a detailed description in combination with specific embodiments, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. The raw materials used in the examples are all commercially available unless otherwise specified.

[0023] Example 1 Preparation of all-male offspring chickens

[0024] 1. Cell collection

[0025] (1) Dissect the chicken embryo, remove the tissues of other parts, strip a pair of gonads from the kidneys, put the gonads into 200 μL of DPBS buffer for washing, and clean the remaining residual tissues; put the separated gonads into a PCR tube containing 50 μL of trypsin, and then put the PCR tube into a 37 °C water bath for digestion for 8 minutes. During this period, take out the PCR tube every two minutes and shake it evenly to make the gonads fully contact with trypsin;

[0026] (2) When the gonad has no complete shape and only small pieces of debris remain, add 50 μL of mKO culture medium to stop digestion. Aspirate the liquid in the PCR tube into a 15 mL centrifuge tube, place the centrifuge tube in a centrifuge, centrifuge at 800 rpm for 5 minutes, aspirate the supernatant and add it to a 24-well plate, and then place it in an incubator for adherent treatment for 6 hours;

[0027] Among them, the mKO culture medium contains the following components: mKO-DMEM (KnomKOut Dulbecco's Modified Eagle Medium) with a mass percentage of 90.4%, chicken serum with a mass percentage of 0.20%, fetal bovine serum with a mass percentage of 2.00%, sodium pyruvate with a mass fraction of 1.2 mM, heparin sodium at 100 μg / mL, penicillin-streptomycin solution (Anti-Anti) with a volume fraction of 1×, glutamine with a volume fraction of 1×, GS nucleotide supplement with a volume fraction of 1×, β-mercaptoethanol with a mass fraction of 0.1 mM, recombinant human basic fibroblast growth factor b (hFGFb) at 4 ng / mL, lysine with a volume fraction of 1×, B-27 vitamin A with a volume fraction of 1×, and human activin A at 25 ng / mL;

[0028] (3) After the adherent treatment is completed, aspirate the culture medium into a 15 mL centrifuge tube, wash the 24-well plate 2 to 3 times with mKO culture medium and also put it into the centrifuge tube, place the centrifuge tube in a centrifuge, centrifuge at 800 rpm for 5 minutes, aspirate the supernatant and add it to the insert in the 24-well plate. Add 800 μL of mKO culture medium outside the insert and 100 - 200 μL of mKO culture medium into the insert. Then, perform the proliferation and passage of primordial germ cells to obtain male primordial cell germ lines ( Figure 1 );

[0029] The sex of the isolated PGCs was identified by PCR technology. The primers used were 5’-TGCAGAAGCAATATTACAAGT-3’ and 5’-AATTCATTATCATCTGGTGG-3’. The electrophoresis results of the PCR products are as Figure 2 shown, Figure 2 in which lanes 1, 2, 3, 4, and 5 are all single-embryo established line PGC samples detected. The amplified product fragment size corresponding to the Z chromosome is 482 bp, and the amplified product fragment size corresponding to the W chromosome is 326 bp. The results show that they are all male PGCs, indicating that male PGCs of single embryos were successfully obtained in this experiment.

[0030] 2. Preparation of chimeric roosters and chimeric hens

[0031] (1) Collect the semen of HSV gene-edited roosters and breed them with wild-type hens, and collect the fertilized eggs;

[0032] (2) The collected hatching eggs are placed in a constant-temperature incubator for 2 days. A small opening is made in the air chamber of the hatching eggs, and male primordial germ cells for single-embryo line establishment are aspirated with an injection needle and injected into the chicken embryo heart. At the same time, ganciclovir is injected into the gene-edited chicken embryos to specifically eliminate endogenous germ cells.

[0033] (3) Seal the gap of the hatching eggs with sealing film, and put the injected hatching eggs back into the incubator until the chicks hatch, obtaining chimeric roosters and chimeric hens.

[0034] Among them, the preparation method of the above HSV gene-edited roosters is as follows: Using the CRISPR / cas9-mediated gene site-directed knock-in technology, the herpes simplex virus thymidine kinase gene (HSV-TK) is targeted and knocked into the 3' end of the DAZL gene specifically expressed in germ cells, and an EGFP fluorescent reporter gene is inserted to observe the primordial germ cells after gene modification, thus successfully preparing HSV-TK gene-edited chickens that can specifically eliminate endogenous germ cells.

[0035] As Figure 3 shown, the primordial germ cells in the HSV gene-edited recipient chicken embryos express green fluorescence. After adding ganciclovir, the endogenous primordial germ cells in the gonads are eliminated. Therefore, the obtained male primordial germ cells are injected into the recipient chicken embryos to obtain chimeric chickens carrying male primordial germ cells.

[0036] 3. Production of all-male offspring

[0037] (1) The hatched chimeric chickens are fed normally until sexual maturity, and the chimeric roosters and hens are managed separately.

[0038] (2) Collect the semen of the chimeric roosters and artificially inseminate the chimeric hens.

[0039] (3) Collect the hatching eggs laid by the chimeric hens and place them in a constant-temperature incubator for hatching until the chicks hatch.

[0040] (4) The hatched chicks are all-male offspring chickens.

[0041] Experimental results:

[0042] As Figure 1 shown, PGCs isolated from a single chicken embryo, and the PGC cell line established after culture.

[0043] As Figure 2 shown, the sex of the isolated PGCs is identified by PCR technology, and the results show that they are all male PGCs. Therefore, we successfully obtain single-embryo male PGCs.

[0044] As Figure 3As shown, the PGCs in the HSV gene-edited recipient chicken embryos expressed green fluorescence. After adding ganciclovir, the endogenous PGCs in the gonads were eliminated. Therefore, the obtained male PGCs were injected into the recipient chicken embryos to obtain chimeric chickens carrying male PGCs.

[0045] The foregoing description of specific exemplary embodiments of the invention has been presented for purposes of illustration and example. It is not intended to limit the invention to the precise forms disclosed, and obviously, many modifications and variations are possible in light of the above teaching. The purpose of selecting and describing exemplary embodiments was to explain the specific principles of the invention and its practical application so as to enable those skilled in the art to make and utilize various different exemplary embodiments of the invention, as well as various different selections and modifications. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A method for preparing all-male offspring chickens, characterized in that, It includes the following steps: Step S1, cell collection: Isolate male primordial germ cells from the gonads of a single 7-day-old hatched chicken embryo and establish a male primordial germ cell line; Step S2, preparation of chimeric roosters and chimeric hens: Inject male primordial germ cells into HSV gene-edited chicken embryos whose endogenous primordial germ cells have been induced to be eliminated by ganciclovir to obtain chimeric roosters and chimeric hens; Step S3, production of all-male offspring: Mate the chimeric roosters with the chimeric hens to obtain all-male offspring chickens.

2. The method according to claim 1, wherein The method of cell collection in Step S1 is: Take out the gonads from a single male chicken embryo, digest the gonads with trypsin, add mKO culture medium, centrifuge and take the supernatant and put it in an incubator for adherent treatment for 6 hours. After the adherent treatment is completed, centrifuge again and take the supernatant for the proliferation and passage of primordial germ cells to establish a male primordial germ cell line.

3. The method according to claim 1, characterized in that, Step S2 includes the following steps: Step S21, collect semen from HSV gene-edited homozygous roosters and breed them with wild-type hens, and collect HSV eggs; Step S22, put the collected eggs into a constant-temperature incubator and incubate for 2 days. Make a small hole in the air chamber of the eggs, inject the male primordial germ cells obtained in Step S1 into the hearts of the gene-edited chicken embryos with a syringe, and at the same time inject ganciclovir into the chicken embryos to specifically eliminate endogenous germ cells; among them, 10,000 male primordial germ cells are injected into each egg; Step S23, seal the notch of the eggs with sealing film, and put the eggs back into the incubator until the chicks hatch out to obtain chimeric roosters and chimeric hens.

4. The method according to claim 3, characterized in that: The preparation method of the HSV gene-edited rooster is: Using the CRISPR / cas9-mediated gene site-directed knock-in technology, target the herpes simplex virus thymidine kinase gene into the 3' end of the DAZL gene specifically expressed in germ cells, and connect an EGFP fluorescent reporter gene to observe the primordial germ cells after gene modification, then the HSV-TK gene-edited chicken that can specifically eliminate endogenous germ cells can be successfully prepared.

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