Method for isolating and culturing chicken primordial germline stem cells and constructing genetically modified chickens

Abstract

The present invention relates to a method for isolating and culturing chicken primordial germline stem cells (PGCs) and constructing genetically modified chickens. Under an optimized culture system, short-term in vitro culture can yield millions of cells and establish stable lines. These lines can also be cultured for over 200 days in vitro while maintaining migration capacity. After genetic modification of the PGCs established in vitro, genetically modified chickens can be obtained via vascular injection. Furthermore, PGCs injected into the blood vessels of chicken embryos can be sorted on the seventh day of incubation, recultured in vitro, and then injected back into the blood vessels of the chicken embryo, thereby improving the efficiency of constructing genetically modified chickens. The present invention also involves freezing PGCs, achieving a resuscitation survival rate exceeding 80%, allowing for long-term storage of isolated or genetically modified PGCs. The present invention has the advantages of high isolation and culture efficiency, and the two-vascular injection method can improve the efficiency of constructing genetically modified chickens. The present invention has promising application prospects in the in vitro isolation, culture, line establishment, cryopreservation, and construction of genetically modified chickens from chicken PGCs.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and agricultural animal genetic breeding and reproduction, and specifically relates to a method for isolating and culturing chicken primordial germline stem cells and constructing gene-modified chickens. Background Art

[0002] Chickens are a vital economic animal, not only satisfying human demand for meat but also providing a rich supply of eggs. With the global population increasing, demand for poultry products is also increasing. However, environmental changes and the prevalence of poultry diseases have severely impacted the development of the poultry industry. Therefore, improving poultry's production traits and enhancing their disease resistance are crucial.

[0003] The most common and powerful methods for mammalian gene editing are pronuclear injection and somatic cell cloning, which can directly produce gene-edited animals. However, due to the specific reproductive characteristics of poultry, it is difficult to edit their genes and pass them on to the next generation using traditional gene editing methods. Compared with mammalian oocytes, avian oocytes have a large cytoplasm that is connected to the yolk, resulting in incomplete cellularization and making pronuclear injection very difficult. In addition, poultry eggs have a high yolk content and poor transparency, making the nucleus of the oocyte difficult to identify, making somatic cell cloning impossible. However, due to the characteristic that avian embryos can develop in vitro, their in vitro development process is easy to observe and manipulate, facilitating cell tracking, and therefore, they are commonly used in various research studies.

[0004] In previous studies, genetic modification of chickens was achieved by injecting viruses or plasmids carrying exogenous genes during the blastoderm stage. Direct injection of exogenous plasmids is very inefficient, while injection of retroviruses carrying exogenous genes is much more efficient. Many transgenic chickens and birds have been successfully created using retroviral injections. However, due to the limited packaging capacity of viruses, large or complex transgenes are difficult to create. Besides viral transgenesis, other traditional methods, such as liposome transfection, are also available, but these are very inefficient. Furthermore, newly laid fertilized eggs have already undergone a period of development, reaching 40,000 to 60,000 cells. Genetic modification at this stage is not only inefficient but also makes reproductive chimerism more difficult. Summary of the Invention

[0005] Based on the current situation that the methods for constructing genetically modified chickens in the prior art are complex and inefficient, the present invention provides a method for isolating and culturing chicken primordial germline stem cells and constructing genetically modified chickens.

[0006] Specifically, the present invention relates to methods for the rapid and efficient isolation, culture establishment, and cryopreservation of chicken primordial germline stem cells (PGCs), as well as methods for constructing genetically modified chickens. These methods can be used for strain conservation and the development of genetically modified chickens.

[0007] Chicken PGCs can be isolated from blood or gonads, and a large number of cells can be obtained by in vitro culture. Since the number of PGCs in the blood is very small, the success rate of establishing a line is very low, and the time for establishing a line is very long. However, a large number of PGCs can be isolated from the gonads, and the optimized culture medium of the present invention can be used to quickly and efficiently establish a line. At the same time, the freezing culture medium can preserve the PGCs for a long time, and a large number of PGCs can be obtained quickly after recovery. Genetically modified chickens can be constructed through these optimized methods, and the method of secondary injection of PGCs can improve the reproductive chimerism ability of PGCs cultured in vitro for a long time, further improving the efficiency of constructing genetically modified chickens.

[0008] The purpose of the present invention can be achieved by the following technical solutions:

[0009] The present invention first provides a PGCs culture medium for culturing chicken primordial germ stem cells. The PGCs culture medium comprises: a basal culture medium of KO-DMEM, 2% (v / v) chicken serum, 20% (v / v) fetal bovine serum, 100× sodium pyruvate (i.e., sodium pyruvate is diluted 100 times to prepare the culture medium, the same below), 100×GlutaMax, 100×NEAA, 1×GS nucleosides supplement, 0.1mM β-mercaptoethanol, 4ng / ml FGF2, and 6ng / ml SCF.

[0010] The present invention further provides a method for rapidly and efficiently isolating and culturing chicken PGCs, comprising the following steps:

[0011] (1) Preparation of chicken embryos: Incubate fertilized eggs in an incubator and use them when they are 5-10 days old;

[0012] (2) Isolation of PGCs: Remove 5-10 day old chicken embryos from the incubator, separate the gonads, and wash them three times with preheated PBS. Transfer a pair of gonads from each chicken embryo into a 1.5 ml centrifuge tube containing 50 μL of 0.05% trypsin. Digest in a 37°C water bath for 8 minutes. Add 1 ml of PGCs culture medium to terminate the digestion, and gently pipette until the gonad tissue blocks become a cell suspension.

[0013] (3) Purification of PGCs: Purify PGCs from the cell suspension using the differential adhesion method;

[0014] (4) Primary culture of PGCs: Culture with PGCs culture medium, change the culture medium every day, subculture every 3-4 days, and replace the feeder layer;

[0015] (5) PGCs subculture and line establishment: The primordial germline stem cells obtained in step (4) were subcultured to a 12-well plate culture medium containing a new feeder layer at a ratio of 1:3 on the 8th day of culture. The culture medium was replaced every day to obtain PGCs after line establishment.

[0016] In one embodiment of the present invention, in step (3), the method for purifying PGCs using the cell suspension using the differential adhesion method is as follows: the cell suspension is transferred to a 12-well plate, placed in an incubator at 37°C, 5% CO2 and cultured for 3 hours, the non-adherent cells are collected and transferred to a 12-well plate covered with a feeder layer for continued culture, and a pair of gonads from each chicken embryo is cultured separately as a group.

[0017] In one embodiment of the present invention, in step (4), the feeder layer is prepared by treating mouse embryonic fibroblasts with 10 μg / ml mitomycin C for 2 hours, and the feeder layer is replaced every 3-4 days during the PGCs culture process;

[0018] The present invention further provides a method for freezing and thawing PGCs after establishment, comprising the following specific steps:

[0019] (1) PGCs freezing: The cultured PGCs were digested with 0.05% trypsin, and the reaction was terminated with PGCs culture medium. The cells were transferred to a 15ml centrifuge tube and centrifuged at 200g for 4 minutes. The supernatant was aspirated and the cells were resuspended with PGCs freezing solution to obtain a cell suspension. 500μl of cell suspension was added to each cryovial. The number of cells frozen in each cryovial was approximately 100,000-200,000. The cryovial was then placed in a freezing box and placed in a -80℃ refrigerator overnight. The next day, the cells were transferred to liquid nitrogen for long-term storage.

[0020] (2) PGCs recovery: Remove the cryovials from liquid nitrogen and thaw them quickly in a 37°C water bath. Then, transfer the thawed cells to a 15 ml centrifuge tube in a biosafety cabinet, add PGCs culture medium, centrifuge, resuspend in PGCs culture medium, and transfer to a 12-well plate with feeders pre-laid for culture.

[0021] In one embodiment of the present invention, the PGCs in step (1) include PGCs isolated from chicken embryo gonads and cultured in vitro for more than half a month, or PGCs cultured in vitro for different time periods, or PGCs amplified in vitro after genetic modification.

[0022] In one embodiment of the present invention, the PGCs freezing solution in step (1) is: 10% (v / v) DMSO, 10% (v / v) FBS, and 80% (v / v) PGCs culture medium.

[0023] The present invention further provides a method for constructing a genetically modified PGCs cell line, comprising the following specific steps:

[0024] PGCs are isolated from chicken embryo gonads and cultured and expanded in vitro. The genes in the PGCs are then genetically modified, including genetic modification, gene knockout, and gene knockin, to obtain a gene-modified PGCs cell line. The gene-modified PGCs are cultured in vitro under the same culture conditions as those of PGCs.

[0025] Regarding the method for constructing a genetically modified PGCs cell line provided by the present invention, the following steps are specifically described using the construction of a tdTomato transgenic PGCs cell line as an example:

[0026] (1) PGCs were isolated from the gonads of chicken embryos and cultured in vitro. When the number of PGCs reached approximately 100,000 to 200,000, they were electroporated using a Lonza electroporator. The electroporated plasmids contained PB-CAG-tdTomato and PB transposase. After electroporation, the cells were cultured in vitro under the same conditions as those for PGCs.

[0027] (2) Flow sorting: The PGCs electroporated in step (1) are cultured in vitro for 8-12 days and then flow sorted to obtain tdTomato-positive PGCs, which are then cultured and expanded in vitro;

[0028] (3) Freeze the expanded tdTomato-positive PGCs.

[0029] The present invention further provides a method for constructing a genetically modified chicken, comprising the following specific steps:

[0030] (1) Genetically modified PGCs were injected into chicken embryos at 50-52 hours of development via peripheral blood injection and continued to incubate. The number of cells injected was 2000-3000 per embryo.

[0031] (2) Identification of chimera chicken embryos: The chicken embryos of step (1) are further incubated, and on the 7th, 10th, and 15th days of incubation, fluorescence or PCR identification is used to determine whether they are chimeras. The specific method is as follows: the chicken embryos of step (1) are further incubated, and on the 7th, 10th, and 15th days of incubation, part of the chicken embryos is taken out, and the gonads are separated to observe the fluorescence; or the gonadal PGCs are separated, digested, and the genome is extracted, primers are designed according to the genes in the gene-modified PGCs cell line, and PCR identification is performed using specific primers to determine whether they are chimeras. If they are chimeras, it indicates that the gene-modified PGCs can be embedded in the reproductive system, and if they are not chimeras, it indicates that the gene-modified PGCs cannot be reproductively chimeric.

[0032] (3) After the hatched chimeric rooster develops to adulthood, semen is collected to extract the genome, and PCR identification or fluorescence observation is performed using the specific primers used when constructing the gene-modified PGCs cell line to confirm that the PGCs cultured in vitro and gene-modified can develop normally into sperm. If gene-modified sperm can be detected, it means that the gene-modified chicken is successfully constructed; otherwise, the gene-modified chicken construction has failed.

[0033] The following is an example of constructing tdTomato transgenic chickens to provide a detailed description of the process. The construction of other genetically modified chickens, including transgenic, gene knockout, and gene knockin, is similar to this method and includes the following specific steps:

[0034] (1) The tdTomato gene-modified PGCs cell line was injected into chicken embryos at 50-52 hours of development via peripheral blood injection and continued to incubate. The number of injected cells was 2000-3000 per embryo.

[0035] (2) Identification of chimeric chicken embryos: The chicken embryos of step (1) are further incubated, and the fluorescence is used to determine whether they are chimeras on the 7th, 10th, and 15th days of incubation. The specific method is as follows: the chicken embryos of step (1) are further incubated, and part of the chicken embryos is taken out on the 7th, 10th, and 15th days of incubation, and the gonads are separated to observe the fluorescence. If there is red fluorescence, it is a chimeric chicken embryo. If it is a chimera, it indicates that the genetically modified PGCs can be embedded in the reproductive system. If it is not a chimera, it indicates that the genetically modified PGCs cannot be reproductively chimeric.

[0036] (3) After the hatched chimeric rooster develops to adulthood, semen is collected and observed for fluorescence to confirm that the PGCs transformed with tdTomato in vitro can normally develop into sperm. If red fluorescent sperm can be detected, it means that the genetically modified chicken is successfully constructed; otherwise, the genetically modified chicken fails to be constructed.

[0037] The present invention further provides a method for constructing a genetically modified chicken based on a method of secondary vascular injection of PGCs, which can improve the efficiency of constructing the genetically modified chicken. The obtained genetically modified PGCs cells are injected into a chicken embryo that has developed for 50-52 hours through a peripheral vascular injection method and continue to be incubated. The amount of injected cells is 2000-3000 per embryo. After the injection, the chicken embryo gonads are separated to obtain PGCs. The genetically modified PGCs are screened and continued to be cultured and amplified in vitro. After amplification to a certain number, the genetically modified PGCs are injected into the chicken embryo that has developed for 50-52 hours through a peripheral vascular injection method for a second time and continue to be incubated, thereby finally obtaining the genetically modified chicken.

[0038] The following uses the method of constructing tdTomato transgenic chickens by secondary intravascular injection as an example to illustrate the specific operation method, which includes the following steps:

[0039] (1) tdTomato-positive PGCs were injected into chicken embryos at 50-52 hours of development via peripheral blood injection and continued to incubate. The amount of injected cells was 2000-3000 per embryo. After injection, the embryos were continued to incubate until the seventh day, and the gonads of the chicken embryos were isolated to obtain PGCs.

[0040] (2) Flow cytometry sorting: The PGCs isolated in step (1) are cultured in vitro for 4-8 days and then flow cytometry sorted to separate tdTomato-positive PGCs, which are then cultured in vitro;

[0041] (3) Secondary vascular injection of PGCs: After the sorted tdTomato-positive PGCs are cultured in vitro to a certain number, they are injected into chicken embryos at 50-52 hours of development via peripheral vascular injection and continued to hatch. Transgenic chickens carrying the tdTomato gene are obtained. The secondary vascular injection of PGCs can improve reproductive chimerism and increase the efficiency of obtaining genetically modified chickens.

[0042] In response to the problems of existing chicken PGCs separation, line establishment, cryopreservation, in vitro culture, construction of genetically modified chickens and reproductive chimera efficiency, the present invention establishes a method for rapid and efficient separation and stable line establishment of chicken embryo gonad PGCs. PGCs can be isolated and purified from the gonads of chicken embryos hatched for 5-10 days. Utilizing an optimized culture medium and cell freezing system, rapid amplification and line establishment and long-term cell freezing can be achieved. The method can also be applied to the construction of genetically modified chickens, and the reproductive chimera ability is improved by a method of secondary chicken embryo vascular injection, thereby improving the efficiency of constructing genetically modified chickens. Experiments have shown that the method of the present invention has a high success rate, a short time, is stable and efficient, and the PGCs of the established line express germline stem cell-specific markers and maintain good gonadal migration ability. Genetically modified chickens can also be constructed, and the method of secondary chicken embryo vascular injection can improve the efficiency of constructing genetically modified chickens.

[0043] The present invention overcomes the shortcomings of the prior art by providing a rapid, short-term, stable, and efficient method for rapidly isolating and establishing a lineage from the gonads of 5-10 day-old chicken embryos, as well as a method for long-term storage of PGCs by freezing in liquid nitrogen. These methods are applied to the preservation of strain resources and the construction of genetically modified chickens. Furthermore, secondary vascular injection of PGCs is used to improve the reproductive chimerism of long-term in vitro culture of PGCs, thereby optimizing the efficiency of constructing genetically modified chickens.

[0044] Genome modification of chicken primordial germline stem cells is more efficient and is a reliable method for constructing genetically modified chickens.

[0045] Specifically, the outstanding advantages of the present invention are:

[0046] (1) The PGCs in the present invention are derived from the gonads of chicken embryos incubated for 5-10 days, which has a long time span, flexible operability, simple operation and good reproducibility;

[0047] (2) The present invention has the characteristics of fast speed, high separation efficiency, and short establishment time, and can expand to millions of cells in vitro after half a month of culture;

[0048] (3) The optimized culture system of the present invention can stably and efficiently establish PGCs cell lines, with a success rate of over 80%. PGCs cultured in vitro for more than 200 days still retain the characteristics of primitive germline stem cells and have good gonadal migration ability;

[0049] (4) The optimized cell freezing formula of the present invention has the advantages of simple preparation, high efficiency, and wide applicability, and the cell survival rate after recovery reaches more than 80%;

[0050] (5) The present invention uses this method to construct a tdTomato-transfected PGCs cell line and construct tdTomato transgenic chickens by chicken embryo vascular injection. Reproductive chimeras and chimeric transgenic chickens can be observed on the 7th, 10th, and 15th days of incubation. More genetically modified chickens will be constructed later.

[0051] (6) The present invention improves reproductive chimerism through the method of secondary chicken embryo vascular injection, so that PGCs that have been cultured in vitro for a long time or screened for a long time after gene editing have better reproductive chimerism, thereby improving the efficiency of constructing genetically modified chickens. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 The present invention is the primordial germ stem cells separated and purified from E7-E10 day chicken embryos;

[0053] Figure 2 The primordial germ stem cells are isolated and purified and cultured in vitro for 16 days according to the present invention;

[0054] Figure 3 The tdTomato gene-transfected primitive germline stem cells established by the present invention;

[0055] Figure 4 The tdTomato transgenic primordial germline stem cell line established by the present invention migrates to the gonads of E7 day chicken embryos and settles thereafter after being injected into blood vessels;

[0056] Figure 5 The tdTomato transgenic primordial germline stem cell line established by the present invention is injected into blood vessels and migrates to the gonads of E7-day chicken embryos to settle, and then tdTomato-positive PGCs are isolated and purified to establish the line;

[0057] Figure 6 The tdTomato transgenic primitive germ stem cell line obtained by separation and purification of the present invention migrates to the gonad of E7 day chicken embryos and settles therein after secondary vascular injection.

[0058] Figure 7 Primordial germline stem cells were identified by SSEA1 staining. DETAILED DESCRIPTION

[0059] The breeding eggs used in the following examples were from Jingbai laying hens.

[0060] Example 1

[0061] Provided is a method for rapidly and efficiently isolating and culturing chicken PGCs, comprising the following steps:

[0062] (1) Hatching of eggs

[0063] Freshly laid Jingbai laying hen eggs were transported back to the laboratory, and the surface was wiped with 75% alcohol. After drying, the eggs were placed in an incubator and incubated at 38°C and 60% humidity. They were taken out on the 5th to 10th day.

[0064] (2) Isolation of PGCs

[0065] Remove 5-10 day-old hatching eggs to a sterile operating room. Wipe the surface of the eggs with 75% alcohol. Use sterile curved forceps to open the large end of the egg and remove the 5-10 day-old chick embryos. Place them in a 100 mm cell culture dish with PBS, wash three times, and transfer them to a new 100 mm dish lid. Dissect the chick embryos under a stereomicroscope. Use fine-pointed forceps to remove the tail, tear open the abdomen, remove the internal organs, and visualize the gonads. Gently scrape the gonads with two fine-pointed forceps and transfer them to a previously prepared 50 μL drop of preheated PBS for washing. Repeat this washing three times. After that, transfer each pair of gonads into a centrifuge tube containing 50 μL of 0.05% trypsin (1.5 mL). Digest in a 37°C waterbath for 8 minutes. Then, open the lid in a biosafety cabinet, add 1 mL of preheated PGCs medium to terminate the digestion, and gently pipette with a 1 mL pipette until the gonadal tissue fragments become a cell suspension.

[0066] (3) Purification of PGCs

[0067] The cell suspension was transferred to a 12-well plate and cultured in a 37°C, 5% CO2 incubator for 3 h. The non-adherent cells ( Figure 1 ) were transferred to a 12-well plate covered with feeder and cultured. Each pair of gonads from the chick embryo was cultured separately as a group.

[0068] (4) Primary culture of PGCs

[0069] Cultured with PGCs medium (KO-DMEM, 2% chicken serum, 20% fetal bovine serum, 100× sodium pyruvate, 100× GlutaMax, 100× NEAA, 1× GS nucleoside supplement, 0.1 mM β-mercaptoethanol, 4 ng / ml FGF2, 6 ng / ml SCF), the medium was changed every day, and the cells were passaged every 3-4 days and the feeder layer was replaced;

[0070] (5) PGCs subculture and line establishment

[0071] On the 8th day of culture, the PGCs were subcultured into a 12-well plate containing a new feeder layer at a ratio of 1:3, and the culture medium was changed every day. When the cells could be stably subcultured in vitro for half a month, the line was successfully established ( Figure 2 ).

[0072] Example 2

[0073] This example further provides a method for freezing and thawing PGCs after the establishment of the line in Example 1, comprising the following specific steps:

[0074] (1) PGCs freezing: The cultured PGCs were digested with 0.05% trypsin, and the reaction was terminated with PGCs culture medium. The cells were transferred to a 15 ml centrifuge tube and centrifuged at 200 g for 4 minutes. The supernatant was aspirated and the cells were resuspended with PGCs freezing solution (10% DMSO, 10% FBS, 80% PGCs culture medium). 500 μl of cell resuspension solution was added to each cryovial. The number of cells frozen in each cryovial was approximately 100,000 to 200,000. The cryovials were then placed in a freezing box and placed in a -80°C refrigerator overnight. The next day, the cells were transferred to liquid nitrogen for long-term storage.

[0075] (2) PGCs recovery: Remove the cryovials from liquid nitrogen and thaw them quickly in a 37°C water bath. Then, transfer the thawed cells to a 15 ml centrifuge tube in a biosafety cabinet, add PGCs culture medium, centrifuge, resuspend in PGCs culture medium, and transfer to a 12-well plate with feeders pre-laid for culture.

[0076] Among them, in the freezing and thawing method provided in this embodiment, the PGCs described in step (1) also include PGCs isolated from chicken embryo gonads and cultured in vitro for more than half a month, or PGCs cultured in vitro for different time periods, or PGCs amplified in vitro after genetic modification.

[0077] Example 3

[0078] This example provides a method for constructing a tdTomato transgenic PGCs cell line, comprising the following specific steps:

[0079] (1) PGCs were isolated from the gonads of seven-day-old chicken embryos and cultured and expanded in vitro. When the number of PGCs reached approximately 100,000 to 200,000, they were electroporated using 6 μg of PB-CAG-tdTomato and 3 μg of PB transposase plasmids. The cells were then cultured in vitro.

[0080] (2) Flow sorting: The PGCs electroporated in step (1) were cultured in vitro for 8-12 days and then flow sorted to obtain tdTomato-positive PGCs, which were then cultured and expanded in vitro ( Figure 3 );

[0081] (3) Freeze the expanded tdTomato-positive PGCs or proceed with the next experiment intermittently.

[0082] Example 4

[0083] This embodiment provides a method for constructing a genetically modified chicken, comprising the following specific steps:

[0084] The PGCs cultured and expanded in vitro were injected into chicken embryos at 50-52 hours of development through peripheral blood injection and continued to incubate. The amount of injected cells was 2000-3000 per embryo. Then, on the 7th, 10th and 15th days of incubation, fluorescence ( Figure 4 ) to determine whether it is a chimera, which refers to the case where the gene modification is fluorescent, and is judged by fluorescence, such as observing red light, green light, etc.; if there is no fluorescence, specific primers are designed through the gene modified and identified by PCR method; after the rooster to be hatched develops to adulthood, semen is collected to extract the genome, and specific primers are used for PCR identification or fluorescence observation to determine that the PGCs cultured in vitro and genetically modified can develop into sperm normally.

[0085] Secondary vascular injection of PGCs:

[0086] Chicken PGCs were injected into chicken embryos at 50-52 hours of development through peripheral blood injection and continued to incubate. The amount of injected cells was 2000-3000 per embryo. On the seventh day of incubation, the chicken embryonic gonads were separated to obtain PGCs, and positive PGCs were screened ( Figure 5 ) Continue to culture and expand in vitro, and after expanding to a certain number, inject the positive PGCs into the chicken embryos of 50-52 hours of development through peripheral blood injection and continue to incubate ( Figure 6 The reproductive chimerism ability of positive PGCs can be improved by secondary injection.

[0087] Identification of PGCs by immunofluorescence staining

[0088] PGCs cultured in vitro were smeared on a glass slide, dried, and fixed with 4% paraformaldehyde. They were then permeabilized with TritonX-100, blocked with BSA, and stained with primary antibodies overnight. The next day, secondary antibodies were used for staining, and the cell nuclei were stained with DAPI. The results showed that the cell membranes of the isolated PGCs expressed the primordial germ cell-specific marker SSEA1, and the cell nuclei were stained with DAPI ( Figure 7 ).

[0089] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. Improvements and modifications made by those skilled in the art based on the disclosure of the present invention, without departing from the scope of the present invention, should be within the scope of protection of the present invention.

Claims

1. A method for constructing genetically modified chickens based on the secondary vascular injection of PGCs, characterized in that: The following steps are involved: Construction of gene-modified PGCs cell lines; The obtained gene-modified PGCs cell line is injected into a chicken embryo at 50-52 hours of development via peripheral vascular injection and continued to be incubated. After the injection, the chicken embryo gonads are separated to obtain PGCs. The gene-modified PGCs are screened and further cultured and amplified in vitro. After amplification to a certain number, the gene-modified PGCs are injected into a chicken embryo at 50-52 hours of development via peripheral vascular injection for a second time and continued to be incubated, thereby finally obtaining a gene-modified chicken. The method for constructing a genetically modified PGCs cell line comprises the following steps: (1) Preparation of chicken embryos: Incubate the fertilized eggs in an incubator and use them after 5-10 days of incubation; (2) Isolation of PGCs: Remove 5-10 day old chicken embryos from the incubator, separate the gonads, wash with preheated PBS, and digest a pair of gonads from each chicken embryo as a group with 0.05% trypsin at 37°C for 8 minutes. Then, add PGCs culture medium and pipette until the gonad tissue blocks become a cell suspension. (3) Purify PGCs from the cell suspension using the differential adhesion method; (4) Primary culture of PGCs: Culture with PGCs culture medium, change the culture medium every day, subculture every 3-4 days, and replace the feeder layer; (5) PGCs subculture and line establishment: The primordial germline stem cells obtained in step (4) were subcultured to a 12-well plate culture medium containing a new feeder layer at a ratio of 1:3 on the 8th day of culture. The culture medium was replaced every day to obtain PGCs after line establishment. PGCs are isolated from chicken embryo gonads and cultured and expanded in vitro, and then genes in the PGCs are genetically modified, including gene modification, gene knockout, and gene knockin, to obtain a gene-modified PGCs cell line, wherein the culture conditions are the same as those of the PGCs; The PGCs culture medium consists of KO-DMEM, 2% (v / v) chicken serum, 20% (v / v) fetal bovine serum, 100 Sodium pyruvate, 100 GlutaMax, 100 NEAA, 1 GS nucleosides supplement, 0.1 mM β-mercaptoethanol, 4 ng / ml FGF2, 6 ng / ml SCF.

2. The method for constructing genetically modified chickens based on the PGCs secondary vascular injection method according to claim 1, characterized in that: In step (3), the method for purifying PGCs using the differential adhesion method of the cell suspension is as follows: the cell suspension is transferred to a 12-well plate, placed in an incubator at 37°C and 5% CO2 for culture, the non-adherent cells are collected and transferred to a 12-well plate covered with feeders for further culture, and a pair of gonads from each chicken embryo is cultured separately as a group.

3. The method for constructing genetically modified chickens based on the PGCs secondary vascular injection method according to claim 1, characterized in that: In step (4), the feeder layer is prepared by treating mouse fetal fibroblasts with 10 μg / ml mitomycin C for 2 h. The feeder layer is replaced every 3-4 days during the PGCs culture process.

4. The method for constructing genetically modified chickens based on the PGCs secondary vascular injection method according to claim 1, characterized in that: During the peripheral blood vessel injection, the amount of injected cells is 2000 to 3000 per embryo.

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