STUDIES ON DERIVATION AND CULTURE OF EMBRYONIC STEM CELLS

点击翻译按钮获取中文摘要

Embryonic stem (ES) cells have been widely used as a powerful tool for the study of cell differentiation and gene targeting, but the techniques for the derivation and of stem cells from embryos are largely of empirical nature and little is known about the factors that influence their isolation. This research was undertaken to establish our own ES cell lines through the improvement of technologies related to the derivation, culture and making chimeras. Two ES cell lines have so far been established from the blastocysts of 129/SvJ mice. The cells from one cell line (A3-1) have been extensively characterized for their pluripotency, including the ability to develop to germ line chimeras following injection into the host blastocysts and transfer to the pseudopregnant recipients. The cells are homozygous for albino locus (c/c genorype) judging from the coat color of the offspring. Pluripotency of A3-1 cells has been further demonstrated following gene targeting. To disrupt the mouse endothelin-1(ET-1) gene, A3-1 cells were electroporated with a linealized targeting vector and clones resistant to G418 and gancyclovir were screened by PCR and Southern blot analysis. The analysis revealed that 35 of 281 G418/gancyclovir-resistant clones were targeted for the TE-1 locus. Breeding of the chimeric males derived from a targeted clone (C31) resulted in germ line transmission in two of 9 males. PCR and Southern analysis of tail DNA revealed that both of them transmitted the mutant ET-1 allele to ES cell-derived progeny in the Mendellian ratio of approximately 50% It can be concluded from these results that the basic technologies for the derivation of ES cells are now available for routine laboratory work.