Reddy S, Rayburn H, von Melchner H, Ruley H E
Center for Cancer Research, Massachusetts Institute of Technology, Cambridge 02139.
Proc Natl Acad Sci U S A. 1992 Aug 1;89(15):6721-5. doi: 10.1073/pnas.89.15.6721.
Murine embryonic stem (ES) cells were infected with a retrovirus promoter trap vector, and clones expressing lacZ fusion genes (LacZ+) were isolated by fluorescence-activated cell sorting (FACS). Of 12 fusion genes tested, 1 was repressed when ES cells were allowed to differentiate in vitro. Two of three lacZ fusion genes tested were passed into the germ line, indicating that FACS does not significantly affect stem cell totipotency. The pattern of lacZ expression observed in vivo was consistent with that seen in vitro. Both fusion genes were expressed in preimplantation blastulas. However, a fusion gene whose expression was unaffected by in vitro differentiation was ubiquitously expressed in day-10 embryos, while the other, which showed regulated expression in vitro, was restricted to cells located along the posterior neural fold, the optic chiasm, and within the fourth ventricle. These results demonstrate the utility of using promoter trap vectors in conjunction with fluorescence sorting to disrupt developmentally regulated genes in mice.
用逆转录病毒启动子捕获载体感染小鼠胚胎干细胞（ES细胞），通过荧光激活细胞分选（FACS）分离出表达lacZ融合基因的克隆（LacZ+）。在测试的12个融合基因中，当ES细胞在体外分化时，有1个基因受到抑制。测试的3个lacZ融合基因中有2个进入了种系，这表明FACS不会显著影响干细胞的全能性。体内观察到的lacZ表达模式与体外观察到的一致。两个融合基因在植入前囊胚中均有表达。然而，一个其表达不受体外分化影响的融合基因在第10天胚胎中普遍表达，而另一个在体外显示出受调控表达的基因则局限于沿后神经褶、视交叉以及第四脑室内的细胞。这些结果证明了将启动子捕获载体与荧光分选结合使用来破坏小鼠发育调控基因的实用性。
