Characterisation and germline transmission of cultured avian primordial germ cells.
BACKGROUND
Avian primordial germ cells (PGCs) have significant potential to be used as a cell-based system for the study and preservation of avian germplasm, and the genetic modification of the avian genome. It was previously reported that PGCs from chicken embryos can be propagated in culture and contribute to the germ cell lineage of host birds.


RESULTS
We confirm these results by demonstrating that PGCs from a different layer breed of chickens can be propagated for extended periods in vitro. We demonstrate that intracellular signalling through PI3K and MEK is necessary for PGC growth. We carried out an initial characterisation of these cells. We find that cultured PGCs contain large lipid vacuoles, are glycogen rich, and express the stem cell marker, SSEA-1. These cells also express the germ cell-specific proteins CVH and CDH. Unexpectedly, using RT-PCR we show that cultured PGCs express the pluripotency genes c-Myc, cKlf4, cPouV, cSox2, and cNanog. Finally, we demonstrate that the cultured PGCs will migrate to and colonise the forming gonad of host embryos. Male PGCs will colonise the female gonad and enter meiosis, but are lost from the gonad during sexual development. In male hosts, cultured PGCs form functional gametes as demonstrated by the generation of viable offspring.


CONCLUSIONS
The establishment of in vitro cultures of germline competent avian PGCs offers a unique system for the study of early germ cell differentiation and also a comparative system for mammalian germ cell development. Primary PGC lines will form the basis of an alternative technique for the preservation of avian germplasm and will be a valuable tool for transgenic technology, with both research and industrial applications.
The Roslin Institute and Royal Dick School of Veterinary Studies, University of Edinburgh, Roslin, United Kingdom.
Joni
J
Macdonald
James D
JD
Glover
Lorna
L
Taylor
Helen M
HM
Sang
Michael J
MJ
McGrew
D000818:Animals; D030801:Animals, Genetically Modified; D001549:Benzamides / Q000494:pharmacology; D019070:Cell Lineage; D049109:Cell Proliferation; D002478:Cells, Cultured; D002642:Chick Embryo; D004159:Diphenylamine / Q000031:analogs & derivatives / Q000494:pharmacology; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D005260:Female; D018507:Gene Expression Regulation, Developmental; D005854:Germ Cells / Q000166:cytology* / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D020935:MAP Kinase Signaling System / Q000187:drug effects; D008297:Male; D008540:Meiosis; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D001549:Benzamides; D004159:Diphenylamine; D049452:Green Fluorescent Proteins; D019869:Phosphatidylinositol 3-Kinases; C506614:mirdametinib