Bernet-Grandaud A, Ouazana R, Morlé F, Godet J
CNRS UMR 106, Université Claude-Bernard, Villeurbanne, France.
C R Acad Sci III. 1994 Oct;317(10):921-9.
We replaced the 3' flanking region of the human alpha 1-globin gene that binds in vitro the specific transcription factors GATA-1 and AP1/NF-E2, by a neo marker gene using homologous recombination in a MEL (mouse erythroleukemia line) hybrid cell line harbouring a single human chromosome 16. Using an improved method of the neo-positive and HSV-tk negative selection, one correctly targeted clone was obtained out of 164 clones analyzed. In contrast to non-targeted clones, the expression of teh neo gene in the targeted clone acquired the erythroid differentiation-dependent inducibility normally characteristic of the alpha-globin genes. No difference was observed in the expression of the human zeta, alpha 2, alpha 1, or theta-globin genes before and after induction of differentiation between the targeted clone and parental cells. These results indicate that, at least in the experimental system used, the 3' flanking region of the human alpha 1-globin gene can be replaced by an exogenous non-erythroid gene without affecting the regulation of the globin genes contained in the alpha-globin cluster.
我们利用同源重组技术，在一个携带单条人类16号染色体的MEL（小鼠红白血病细胞系）杂交细胞系中，将人α1-珠蛋白基因的3'侧翼区域（该区域在体外可结合特异性转录因子GATA-1和AP1/NF-E2）替换为新霉素标记基因。使用改进的新霉素阳性和单纯疱疹病毒胸苷激酶阴性选择方法，在分析的164个克隆中获得了一个正确靶向的克隆。与非靶向克隆不同，靶向克隆中新霉素基因的表达获得了α-珠蛋白基因通常具有的依赖于红系分化的诱导性。在诱导分化前后，靶向克隆与亲本细胞之间的人ζ、α2、α1或θ-珠蛋白基因表达未观察到差异。这些结果表明，至少在所使用的实验系统中，人α1-珠蛋白基因的3'侧翼区域可以被外源非红系基因替换，而不影响α-珠蛋白基因簇中所含珠蛋白基因的调控。
