Study on isolation and culture modes of mouse primordial germ cell and its biological characteristics / 重庆医学

ABSTRACT

Objective To explore a simple and efficient method for isolating and culturing mouse primordial germ cells (mPGCs) in vitro in order to provide the sufficient mPGCs sources of mouse model .Methods The 12 .5 dpc mouse embryonic gen-ital ridge were isolated in vitro and cut into pieces ,then the organization to adherent culture was performed in a-MEM medium con-taining fetal calf serum and insulin-transferrin-selenium (ITS) and follicle stimulating hormone(FSH) and recombinant endothelial growth factor(rEGF) .The inverted microscope was used to observe the cellular morphology .Flow cytometry was used to identify the stage specific embryonic antigens of cultured cells .Reverse transcription-polymerase chain reaction(RT-PCR) and immunofluo-rescence were used to identify the pluripotency gene Oct4 expressions of stem cells and specific genes in the meiosis phaseⅠ .Results The in vitro isolated and cultured mPGCs showed good cell morphology ,extremely strong proliferation capacity and the positive expression of SSEA-1 and negative expression of SSEA-4 ,Oct4 ,meanwhile the expressions of Stra8 ,Vasa ,Scp3 ,Zp3 were detected to be positive .Conclusion Using this culture method can culture the high purity of mPGCs with the excellent stem cell properties and extremely strong proliferative ability ,moreover which makes the cells entering the meiosis stage .