| The production of transgenic chickens through chimeric intermediates requires the incorporation of stable genetic modification into chicken blastodermal cells (CBCs). Transfected CBCs were isolated using antibiotic resistance and magnetic activated cell sorting (MACS).; CBCs that were lipofectin transfected with linearized pZeoSV2/ lacZ were selected in the presence of 375 mug Zeocin(TM) per ml medium for 6 days. About 450 resistant colonies were obtained from 1.5 x 105 transfected cells. The presence of the transgene in the resistant colonies was detected by PCR amplification with LacZ, Zeo or forward Zeo and reverse LacZ primers. Staining with X-gal and the fluorescent substrate 5-dodecanoylaminofluorescein di-beta-D-galactopyranoside (C 12FDG) revealed expression of the bacterial lacZ gene in 10% and 70% of the resistant colonies respectively. Following the injection of these resistant cells, no contribution of the selected donor cells was found in recipient embryos.; CBCs co-electroporated with pmiwZ and pMACS K k were sorted with MACS based on expression of the mouse H-2K k molecule on the surface of CBCs which were cultured for 24 and/or 48 hour. Sorted cells contributed to both extra-embryonic and intra-embryonic tissues in 72 hour recipient embryos. Furthermore, the MACS procedure enriched the transfected CBCs which had been maintained in culture for 48 hours and the injection of the enriched cells marginally increased the level of intra-embryonic incorporation. To improve the efficiency of expression of H-2Kk epitope on the surface of transfected CBCs, pMiw-H-2Kk, which contains the mouse H-2Kk gene under the control of chicken beta-actin promoter was constructed. The construction was successful as verified by restriction enzyme digestion and improved expression in transfected CHO cells. However, no expression was detected in CBCs that were transfected with pMiw-H-2K k, suggesting that the mechanism regulating expression of H-2K k gene in CBCs is very complex.; In conclusion, by combining the use of a new construct co-expressing a selectable marker and a gene of interest with the mariner transposable element, it is likely that the chances of obtaining a homogenous population of stable transfected CBCs will be increased. |
