Study On The Optimization Of Culture Conditions Of Rabbit Primordial Germ Cells In Vitro

Animal primordial germ cells are ideal models for studying the early developmental mechanism of embryos and related regulatory factors,and their research has become one of the most dynamic,the most popular and cutting-edge issues in field of life science.Animal PGCs because of its infinite proliferation,multi-directional differentiation ability and genetic operability as cell research and gene therapy ideal carrier.This study 14-18 d fetal rabbit as experimental object.The PGCs were isolated and cultured in vitro and to observe the morphological changes of PGCs and colony forming process,using the AKP staining and molecular biological methods to identify rabbit PGCs,and to explore the effects of feeder layer type,serum content and cytokines on the isolation and culture of rabbit PGCs,thus lay the foundation for the construction of rabbit EG cell line.1.In this study,Kunming white inbred mice 13-15 d gestational age fetal mice and Japanese big ear white rabbits 18d-21 d gestational rabbits embryonic fibroblasts were cultured in vitro.And making growth curve,calculate the cell recovery rate of different generations of cells after cryopreservation at different times.Concluded as follow:(1)Mouse,rabbit P3 embryonic fibroblasts strong growth,less hybrid cells,biological traits stable,can be used as seed cells for experimental research;(2)Short-term frozen(30d,60d)mouse and rabbit P1,P3 embryonic fibroblasts after resuscitation of its proliferation and biological characteristics have not been affected.Choose well-grown and purified mouse,rabbit P3 embryonic fibroblasts to freeze for a short time to meet the demand of future experiment.2.This experiment selected 14-18 d gestational Japanese big ear white rabbits as the object to peel genital ridge,PGCs isolated and cultured in vitro.Used enzyme histochemical methods to detect alkaline phosphatase activity and RT-PCR detection of transcription factor expression of Oct-4 to identified PGCs.Inoculated PGCs to mouse,rabbit embryo fibroblast feeder layer made of different density to determine the optimum culture conditions of the species and density.The culture medium,which is most suitable for PGCs breeding,was screened by adding cytokine LIF and adjusting the serum concentration.The conclusion as below:(1)When the primordial germ cells were isolated and cultured in rabbit embryos at 14-18 days.(2)Rabbit primitive germ cells were seeded on the growth layer with 6×104 feeder layer made of rabbit embryonic fibroblasts,the colony morphology was the best and the differentiation rate was slower.This is the optimum condition for the feeder layer.(3)The optimum proportion of the culture medium is: DMEM,1% NEAA Penicillin-Streptomycin Solution,2mmol/L L-Glutamine +0.1mmol/L 2-Mercaptoethano,20ng/m L LIF,15% FBS.According to this ratio broth made to culture rabbit primordial germ cells,more cells can be obtained colonies,and the colonies remain undifferentiated longer.