Selection of genetically modified chicken blastodermal cells by magnetic-activated cell sorting.
The use of chicken blastodermal cells (CBC) in the production of transgenic chickens requires incorporation of the desired stable genetic modification into CBC. With greater proportions of stably transfected blastodermal cells in the embryo inoculum, the frequency of intermediate chimeric birds is greater. Magnetic-activated cell sorting (MACS) was evaluated as a method for enrichment of transfected CBC. This approach requires surface expression of a molecule that can be recognized by an antibody. Chicken blastodermal cells from fertilized Barred Plymouth Rock eggs were coelectroporated with pmiwZ and pMACS Kk and were sorted magnetically by expression of the mouse H-2Kk molecule on the surface of successfully transfected cells. The effectiveness of sorting was assessed using X-gal staining to detect lacZ expression from the pmiwZ plasmid. After 48 h of culture, lacZ-positive cells appeared to be enriched 1.4-fold in the MACS selected population. Cells from this enriched pool contributed to extra-embryonic and intra-embryonic tissues of 72-h White Leghorn recipient embryos with a marginal increase in levels of intra-embryonic contribution. Our demonstration that transfected, cultured, and magnetically sorted CBC maintain their ability to contribute to ectodermal and mesodermal lineages of intra-embryonic tissues illustrates the potential value of this technique for introducing genetic modifications into birds.
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Department of Biomedical Sciences, University of Guelph, Ontario, Canada. wqingxia@hotmail.com
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D000818:Animals; D030801:Animals, Genetically Modified / Q000196:embryology*; D001756:Blastoderm / Q000166:cytology*; D002469:Cell Separation / Q000662:veterinary; D002478:Cells, Cultured; D002642:Chick Embryo / Q000166:cytology*; D002645:Chickens / Q000235:genetics*; D018274:Electroporation / Q000662:veterinary; D005434:Flow Cytometry / Q000662:veterinary; D018189:Immunomagnetic Separation / Q000662:veterinary; D014162:Transfection / Q000379:methods / Q000662:veterinary*