Lentiviral Vector Transfection Of Chicken Embryo Primordial Germ Cells In Vitro And Production Of Germline Chimeras

The use of gonadal primordial germ cells (PGCs) in transgenic study for effective production of germline chimera has unlimited value in the field of animal biotechnology. This tool has been thought to be a very attractive approach: PGCs can be collected from the germinal crescent and the embryonic blood. In previous studies, large numbers of PGCs could be retrieved from the embryonic gonads. Moreover, the PGCs could be successfully cultured in vitro for more than several months.And PGCs are more convenient to manipulate and they can finally differentiate to either spermatogonia in males or oogonia in females. Genetically modified chicken primordial germ cells (PGCs), reintroduced these into chick embryos and then transmitted of the transgene through the germ line of modified birds.Primordial germ cells (PGCs) were isolated from the gonads of ISA brown chicken (Gallus domesticus) embryos at stage 28. PGCs were purified by Ficoll density gradient centrifugation. The purity of PGCs could be up to 70% by identification based on periodic acid-Schiff (PAS) and alkaline phosphatase (AKP) staining. PGCs were co-cultured with gonadal stroma cells in DMEM supplemented with growth factors. A lentiviral vector pLenti6-eGFP was constructed and harvested the virus by cotransfecting 293FT cells with the vector and packaging plasmids.Lentiviruses after concentration were used to transfect chicken PGCs, the transfection efficiency of PGCs was up to 24.19%. For PGC transfer, a small window was made on the sharp end of the recipient egg, and approximately 3-4μl (200cells/μl) of cell suspension was injected into the recipient embryo using a micropipette. The egg window of the recipient embryo was sealed twice with paraffin film and then laid down with the pointed end at the bottom, further incubated for an additional 5.5 days. PCR analysis of DNA from 42 recipient embryos showed that exogenous DNA was detected only in the gonads of two chicks but not the muscle and heart.Our technology will be a supplement of current approaches to transgenesis in poultry and also suggests that manipulation of PGCs may allow a novel approach to genetic modification of other vertebrates.