Germline chimeric chickens from FACS-sorted donor cells

Germline chimeric chickens can be constructed by injecting donor chicken blastodermal cells (CBCs) into recipient embryos and incubating to hatch. Transgenic chickens can be produced through chimeric intermediates if the donor cells are genetically manipulated; the chance of producing a transgenic chimera would be increased by enriching the donor population in transfected cells. To demonstrate that donor CBCs can be sorted according to the expression of a foreign gene, CBCs in suspension were subjected to transfection with plasmid DNA encoding bacterial β-galactosidase (β-gal). Following an overnight incubation, the CBCs were loaded with 5-dodecanoylaminofluorescein di-β-D-galactopyranoside (C12FDG), which is fluorescent after cleavage by β-gal. The treated cells were subjected to fluorescence activated cell sorting (FACS) to give “positive” (fluorescent) and “negative” (non-fluorescent) populations. Almost 100% of the “positive” population showed β-gal activity. “Positive” cells were cultured on mouse SNL 76/7 fibroblast feeder cells and formed colonies, most of which still stained positively for β-gal activity after three days. FACS-sorted cells of Barred Plymouth Rock origin were injected into recipient White Leghorn embryos, resulting in chimeric embryos. Of the 298 embryos injected with sorted cells, 23 (8%; 18 injected with “positive cells, five with “negative”) survived to rearing. Somatic chimerism was seen in 12 of 18 (67%) “positive” and three of five (60%) “negative” birds with the proportion of black pigmentation averaging 19% overall. Twenty birds reached sexual maturity, of which 12 (60%) were somatically chimeric; seven (35%) of these produced donor-derived chicks. Donor CBCs can, therefore, be sorted by FACS according to the expression of a selectable marker gene without impairing their ability to contribute to germline chimeras; this procedure could be incorporated into a practicable method by which to increase the chances of producing a transgenic chicken. Mol. Reprod. Dev. 52:33–42, 1999. © 1999 Wiley-Liss, Inc.