| Primordial Germ Cell(PGC)is a kind of reproductive stem cell,which has the potential to differentiate into mature germ cell and plays an extremely important role in the research of reproductive genetics.In terms of application,PGC has advantages in local chicken conservation and is at the core of genetic manipulation in chickens.The separation and purification methods for PGC are constantly being developed and improved,but the commonly used separation methods are cumbersome,require a lot of reagents,and consume a long time,which may lead to PGC damage and decreased vitality.In order to simplify the steps of PGC purification,reduce damage to PGC during operation,and preserve PGC activity,this study developed a method for separating and purifying PGC using differential adhesion method.Based on this,the purified PGC was prepared for chimeric chicken and gene manipulation.The following results were obtained in this study:1.Digestion of fresh chicken embryonic gonads with a development time of 5.5 days and 7.5 days was performed to obtain a control group with PGC ratios of 2.5% and 3.17% that were not purified by differential adhesion method.Two groups of fresh chicken embryo gonadal cell suspension were subjected to differential adhesion method for 4 and 6 hours,respectively.The proportion of PGC in the cell suspension of the experimental group increased to 21.49%,13.22%,17.38%,and 13.19%,with significant differences(p<0.01).This result indicates that the use of differential adhesion method can significantly increase the content of PGC in the suspension of gonadal digestive cells,thereby achieving the purpose of separation and purification.2.To explore the application of differential adhesion method for separation and purification of PGC,an equatorial window opening method was developed and improved for microinjection of chicken embryos,with an incubation rate of 42.62%.The combination of differential adhesion method for separation and purification of PGC and equatorial window opening method was applied.Wuhei chickens were selected as PGC donors and White-Laihang chickens were selected as recipients.11 chimeric chicks with feather color were obtained,with a chimeric chicken proportion of 11.96%.The above results indicate that the differential adhesion method for separating and purifying PGC can be used for the efficient preparation of chimeric chickens,laying the foundation for the subsequent use of it for the conservation of Wuhei chickens.3.In the process of separation and purification of PGC by differential attachment,the transfection efficiency of PGC by adenovirus vector expressing green fluorescent protein and Lentivirus vector was compared.It was found that adenovirus vector could achieve 31.07%transfection efficiency in PGC,significantly higher than 9.19% transfection efficiency of Lentivirus vector.This result shows that PGC can be separated and purified by differential attachment,and PGC gene can be operated at the same time.It preliminarily verifies the feasibility of differential attachment method in isolation and purification of PGC transgenic and genome editing chickens.It provides an idea for further preparation of transgenic and genome editing chicken by this method.In summary,this study found that the differential adhesion method can easily and quickly isolate and purify PGC,and preliminarily verified that this method can be applied to the preparation of chimeric chickens and gene manipulation of PGC.This study lays the foundation for better utilizing PGC for breed protection and the preparation of transgenic and gene operated chickens. |
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