Targeted gene replacement at the endogenous APRT locus in CHO cells.
Targeted gene replacement at the endogenous APRT locus in CHO cells.
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CHO 细胞内源 APRT 基因座的靶向基因替换。
DOI：
10.1007/bf01233193
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发表时间：
1990
期刊：
影响因子：
--
通讯作者：
Brotherman,KA
中科院分区：
文献类型：
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作者：
Adair,GM;Nairn,RS;Wilson,JH;Scheerer,JB;Brotherman,KA
我们证明了在培养的哺乳动物细胞中对内源的染色体基因座进行靶向基因替换的可行性，采用了类似于酵母遗传操作的常规方法的两步策略。利用通过在CHO APRT基因座定向整合质粒序列(包括GPT基因的功能拷贝)而产生的APRT+重组体，我们已经能够选择通过在靶向整合部位的APRT直接重复之间的染色体内重组而出现的GPT−“突出型”重组体。以每代细胞6.3x10≈6的速率发生互换，导致整合的质粒/gpt基因序列以6.3x10−6的速率出现。根据交叉部位的不同，这种“弹出”事件会导致原始aprt靶基因序列的替换或恢复。
We demonstrate the feasibility of targeted gene replacement at an endogenous, chromosomal gene locus in cultured mammalian cells, employing a two-step strategy similar to an approach routinely used for genetic manipulation in yeast. Utilizing an APRT+recombinant generated by targeted integration of plasmid sequences (including a functional copy of the gpt gene) at the CHO APRT locus, we have been able to select gpt−“pop-out” recombinants that have arisen by intrachromosomal recombination between APRT direct repeats at the targeted integration site. Reciprocal exchanges leading to “pop-out” of integrated plasmid/gpt gene sequences occur at a rate of ≈6.3×10−6per cell generation. Depending on the site of crossover, such “pop-out” events result in either replacement or restoration of the original APRT target gene sequence.