Long term culture, cryopreservation and genetic modification of chicken primordial germ cells

Avian primordial germ cells (PGCs) are precursor of gametes and appear during early stages of embryonic development. Under appropriate culture conditions, these cells can keep their germ cells properties in vitro and are foreseen as promising tools for developing efficient avian genetic engineering and preservation of germplasm. We propose original methods that allow long term expansion, efficient cryopreservation and genetic modification of primary cultures of undifferentiated PGCs. PGCs are collected from embryonic blood during their migratory period and grown in cell-culture insert in the presence of feeder cells (BRL). This physically separated co-culture system along with selective culture medium promoted emergence, selection and proliferation of PGCs lines. Forty percent of blood samples gave rise to lines originating from three commercial layer and two Belgian endangered breeds. PGCs lines were characterized for the expression of the stem cells and PGCs marker SSEA-1 by FACS. RT-PCR confirmed expression of germ-line specific markers (CVH, CDH, DAZL), pluripotency markers (cPouV, cSox2, cNanog), telomerase and CXCR4 receptor. All lines were male although isolated from pooled male and female blood samples. Two cryopreservation methods were developed based upon slow-freezing and aseptic vitrification. Both have shown a similar effectiveness in allowing storage without phenotype drift. Stably expressing lines were obtained by Lipofectamine® mediated transfection of a GFP plasmid. PGCs were subsequently injected in recipient embryos. Persistence of exogenous PGCs in the developing gonad of recipient embryos confirmed that PGCs retain their gonadal colonisation ability, both after long-term culture and after cryopreservation.