Chimeric pigs following blastocyst injection of transgenic porcine primordial germ cells

Abstract

Porcine primordial germ cell (PGC) derived cell lines of WAPhGH-transgenic pigs have been established that were able to contribute to chimeras. PGCs were isolated from day 25 to 28 genital ridges of more than 30 individual transgenic 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 proliferated in an undifferentiated state up to passage 13; two lines were maintained for more than 23 passages. Cell staining experiments for differentiation markers in several cell lines, indicated the presence of pluripotent cells in prolonged cultures. Further characterization using karyotyping revealed a normal, euploid set of chromosomes in cells of passages 15 and higher. Pluripotency of freshly isolated, short-term (up to 24 hr before injection) and long-term cultured, frozen/thawed cells was tested by injection into day 6 recipient blastocysts to give rise to chimeric piglets. The injected embryos (n = 209) were endoscopically transferred into the uterine horns of 11 recipient gilts. Tissue analysis from 49 fetuses and eighteen liveborn piglets for PGC contribution in chimeras was carried out using PCR analysis for the presence of the marker transgene. Thirty-two fetuses showed detectable 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.

Generation of chimeric piglets by injection of embryonic germ cells from inbred Wuzhishan miniature pigs into blastocysts

“…Chimeric piglets derived from pig EG cells have been reported by several groups . In this study, we also confirmed the ability of EG cells to form chimeras.…”

“…We identified that two piglets of eight were chimeras, as indicated by skin pigmentation and DNA analysis. Our results showed a higher contribution of the cultured EG cells in the dead chimera than some reports , that is, the dead chimera showed detectable chimerism in up to five of 13 tissues analyzed: EG cells contributed to tissues derived from all three germ layers, including skin, brain, pancreas, thyroid gland, and blood. More work should be performed about the alive chimeric pig to determine the pluripotency of the EG cells.…”

“…bFGF in the presence of LIF and SCF has been shown to dramatically increase PGC numbers and promote their long‐term proliferation in mouse . It has been shown that pig EG cells can proliferate without those factors during the initial culture period and in long‐term culture on a STO feeder layer, suggesting that growth factors provided by either the STO feeder layer or the cultured PGC themselves were sufficient to support in vitro survival and proliferation of porcine PGC . In our study, considering the long‐term culture period, we added all three factors to the medium for pig EG cell cultures in order to keep the pluripotency of EG cells .…”

“…had obtained PGCs from Hampshire × Yorkshire . Mueller generated EG cell lines using Duroc and German Landrace sows mated with German Landrace boars . Piedrahita et al.…”

Strategies for the Derivation of Pluripotent Cells from Farm Animals

“…1999; Vackova et al. 2007) nor primordial germ cells (Mueller et al. 1999) have been isolated in domestic species (Cibelli et al.…”