Progress toward the culture and transformation of chicken blastodermal cells.
Chicken blastodermal cells can be cultured for short periods of time and retain the ability to contribute to somatic and germline tissues when injected into gamma-irradiated stage X embryos. Such a method has yet to yield a germline transgenic bird, in part due to the low rate of transgene integration into the avian genome. In addition, the short culture period precludes the identification and expansion of those cells that carry an integrated transgene. In this study, two methods were developed that produced blastodermal cells isolated from stage X Barred Plymouth Rock embryos bearing an integrated transgene. Addition of chick embryo extract to the culture medium enabled expansion of single colonies for multiple passages. Southern blot analysis indicated that the transgenes had integrated as a single copy in most of the clones. Cells from passaged, transgenic embryo cells were injected into irradiated stage X White Leghorn embryos, producing hatched chicks that bore the donor cells in their somatic tissues. Transgene sequences were detected in sperm DNA; however, breeding of chimeras did not result in germline transmission of the transgene, indicating that the contribution of the transgenic cells to the germline was either nonexistent or very low.
Wang
Youliang
Y
AviGenics, Inc., Georgia BioBusiness Center, Athens, 30605, USA.
Brooks
Carrie F
CF
Jones
Steven A
SA
Olliff
Lyn K
LK
Morgan
Michael
M
Speksnijder
Gordon L
GL
Foley
Chuck
C
Harvey
Alex J
AJ
D000818:Animals; D001756:Blastoderm / Q000166:cytology* / Q000528:radiation effects*; D001757:Blastomeres / Q000502:physiology / Q000528:radiation effects; D018929:Cell Culture Techniques / Q000639:trends*; D002642:Chick Embryo; D018920:Coculture Techniques; D004351:Drug Resistance / Q000235:genetics; D018274:Electroporation; D005347:Fibroblasts / Q000378:metabolism; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D006822:Hybrid Cells; D051379:Mice; D010957:Plasmids / Q000378:metabolism; D011691:Puromycin / Q000009:adverse effects; D014020:Tissue Extracts / Q000494:pharmacology; D014162:Transfection; D019076:Transgenes
D014020:Tissue Extracts; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D011691:Puromycin