Mitani K, Wakamiya M, Hasty P, Graham F L, Bradley A, Caskey C T
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030, USA.
Somat Cell Mol Genet. 1995 Jul;21(4):221-31. doi: 10.1007/BF02255777.
We examined the ability of an E1, E3-defective adenoviral vector to act as a substrate for homologous recombination with chromosomal DNA by including host chromosomal sequence from the mouse Fgr locus that also contained a selectable marker. After infection of mouse embryonic stem cells, stable integration was selected for neomycin resistance and the efficiency of homologous recombination was evaluated. The adenoviral vector was capable of infecting mouse embryonic stem cells efficiently. Between 30-50% of the input virus reached the nuclei after 24 hours of infection. Surprisingly, even without negative selection, 25-40% of the integration resulted from homologous recombination at m.o.i. 10 and 100, although the absolute efficiency of integration was low. Our results suggest that it is possible to modify the structure of an adenoviral vector to achieve a high gene targeting efficiency, resulting in regulated and long-term expression of an introduced gene.
我们通过包含来自小鼠Fgr基因座的宿主染色体序列（该序列也含有一个选择标记），研究了E1、E3缺陷型腺病毒载体作为与染色体DNA进行同源重组底物的能力。在感染小鼠胚胎干细胞后，选择对新霉素具有抗性的稳定整合，并评估同源重组的效率。腺病毒载体能够高效感染小鼠胚胎干细胞。感染24小时后，30%-50%的输入病毒到达细胞核。令人惊讶的是，即使没有阴性选择，在感染复数为10和100时，25%-40%的整合是由同源重组导致的，尽管整合的绝对效率较低。我们的结果表明，有可能对腺病毒载体的结构进行修饰以实现高基因靶向效率，从而导致导入基因的调控和长期表达。
