cPGCs culture: growth pattern in feeder-less culture medium
Fig.1: Clump of cPGCs in feeder-less conditions. Left panel at 40X; Right panel at 100X.
Primordial Germ Cells(PGCs) are extra-embryonic stem cells that populate the embryonic gonad and differentiate to give rise to sperm or ova depending upon the gender. Since PGCs differentiated products transfers genetic information to next generation hence been widely used as a tool to create transgenic organisms for various purposes. There are plethora of instances where transgenic organisms have been generated using modified PGCs either for industrial purposes or for research. We have also utilized this ability of PGCs to answer certain questions pertaining to our sperm mobility project.
We have established feeder-less lines of PGCs from two chicken lines divergently selected for sperm mobility phenotype. In this study we cultured PGCs from 4 day old chicken embryo. PGCs were cultured in modified DMEM media (without feeder layers). DNA and RNA were isolated and analyzed for gender and PGCs specific gene expression. We are now introducing male specific PGCs into busulfan treated recipient embryos to determine whether PGCs genotype determines sperm mobility phenotype.To fully ascertain that sperms from recipient organisms are derived from donor PGCs, donor PGCs are first genetically manipulated to introduce reporter gene by using CRISPR-Cas9, well known genomic scissors. Our preliminary experiments have shown that busulfan treatment leads to 68% embryo survivability, but the hatchability was reduced to 12% after PGCs injection. Currently we are in the process of transfecting and manipulating cPGCs with CRISPR-Cas9 to get tractable PGCs for further analysis.