Isolation and derivation of mouse embryonic germinal cells.
The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos. Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF). Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days. The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state. Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.
Reproductive Genetics and Stem Cell Laboratory, Department of Biological Sciences, Mississippi State University, Mississippi, USA.
Harold
H
Moreno-Ortiz
Clara
C
Esteban-Perez
Wael
W
Badran
Marijo
M
Kent-First
D000818:Animals; D003584:Cytological Techniques / Q000379:methods*; D004622:Embryo, Mammalian / Q000166:cytology*; D053595:Embryonic Stem Cells / Q000166:cytology*; D005260:Female; D005854:Germ Cells / Q000166:cytology*; D006066:Gonads / Q000196:embryology; D051379:Mice; D008810:Mice, Inbred C57BL; D010063:Ovum / Q000166:cytology