STUDIES ON DERIVATION AND CULTURE OF EMBRYONIC STEM CELLS

胚胎干细胞已被广泛用作研究细胞分化和基因靶向的有力工具，但从胚胎中提取和提取干细胞的技术在很大程度上是经验性质的，对影响其分离的因素知之甚少。此次研究是为了通过改良衍生、培养、嵌合体制造等技术，建立自己的胚胎干细胞系而进行的。目前已从129/SvJ小鼠的囊胚中建立了2个胚胎干细胞系。来自一个细胞系（A3-1）的细胞因其多能性而被广泛表征，包括在注射到宿主囊胚和转移到假妊娠受体后发育成生殖系嵌合体的能力。从后代的毛色判断，细胞为纯合的白化位点（c/c基因型）。基因靶向后，A3-1细胞的多能性得到进一步证实。为了破坏小鼠内皮素-1（ET-1）基因，用线性化的靶向载体电穿孔A3-1细胞，通过PCR和Southern blot分析筛选G418和更昔洛韦耐药克隆。分析结果显示，281个G418/更昔洛韦耐药克隆中有35个被定位于TE-1位点。从目标克隆（C31）中获得的嵌合雄性在9只雄性中有两只实现了种系传播。尾巴DNA的PCR和Southern分析显示，它们都以大约50%的孟德尔比率将突变的ET-1等位基因传递给胚胎干细胞衍生的后代。从这些结果可以得出结论，胚胎干细胞衍生的基本技术现在可以用于常规的实验室工作。

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.