Chimeric pigs following blastocyst injection of transgenic porcine primordial germ cells

Porcine primordial germ cell (PGC) derived cell lines of WAPhGH-transgenic pigs have been established that were able to contribute to chimeras. PGCs w ere isolated from day 25 to 28 genital ridges of more than 30 individual tr ansgenic fetuses in order to have an easy to follow marker gene. To support undifferentiated growth, cell lines were derived and stable maintained on STO no. 8 feeder cells, a murine embryonic fibroblast cell line expressing recombinant, membrane-bound porcine stem cell factor (SCF). Fifteen lines p roliferated in an undifferentiated state up to passage 13; two lines were m aintained for more than 23 passages. Cell staining experiments for differen tiation markers in several cell lines, indicated the presence of pluripoten t cells in prolonged cultures. Further characterization using karyotyping r evealed a normal, euploid set of chromosomes in cells of passages 15 and hi gher. Pluripotency of freshly isolated, short-term (up to 24 hr before inje ction) and long-term cultured, frozen/thawed cells was tested by injection into day 6 recipient blastocysts to give rise to chimeric piglets. The inje cted embryos (n = 209) were endoscopically transferred into the uterine hor ns of 11 recipient gilts. Tissue analysis from 49 fetuses and eighteen live born piglets for PGC contribution in chimeras was carried out using PCR ana lysis for the presence of the marker transgene. Thirty-two fetuses showed d etectable chimerism in up to five out of 12 tissues analyzed. Skin samples from eight piglets were positive for the transgene, four of them displayed coat colour chimerism. (C) 1999 Wiley-Liss, Inc.