Adair G M, Nairn R S, Wilson J H, Seidman M M, Brotherman K A, MacKinnon C, Scheerer J B
University of Texas M. D. Anderson Cancer Center, Science Park-Research Division, Smithville 78957.
Proc Natl Acad Sci U S A. 1989 Jun;86(12):4574-8. doi: 10.1073/pnas.86.12.4574.
We have developed a system that permits analysis of targeted homologous recombination at an endogenous, chromosomal gene locus in cultured mammalian cells. Using a hemizygous, adenine phosphoribosyltransferase (APRT)-deficient, Chinese hamster ovary (CHO) cell mutant as a transfection recipient, we have demonstrated correction of a nonrevertible deletion mutation by targeted homologous recombination. Transfection with a plasmid carrying a fragment of the APRT gene yielded APRT+ recombinants at a frequency of approximately 4.1 x 10(-7). The ratio of targeted recombination to nontargeted integrations of plasmid sequences was approximately 1:4000. Analysis of 31 independent APRT+ recombinants revealed conversions of the endogenous APRT gene, targeted integration at the APRT locus, and a third class of events in which the plasmid donor APRT fragment was converted to a full-length, functional gene.
我们开发了一种系统，该系统允许在培养的哺乳动物细胞中的内源性染色体基因位点分析靶向同源重组。使用半合子、腺嘌呤磷酸核糖转移酶（APRT）缺陷的中国仓鼠卵巢（CHO）细胞突变体作为转染受体，我们已经证明通过靶向同源重组可以纠正不可逆的缺失突变。用携带APRT基因片段的质粒转染，产生APRT+重组体的频率约为4.1×10（-7）。质粒序列的靶向重组与非靶向整合的比例约为1:4000。对31个独立的APRT+重组体的分析揭示了内源性APRT基因的转化、在APRT位点的靶向整合，以及第三类事件，即质粒供体APRT片段被转化为全长功能基因。
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