Targeting vector configuration and method of gene transfer influence targeted correction of the APRT gene in Chinese hamster ovary cells.
Targeting vector configuration and method of gene transfer influence targeted correction of the APRT gene in Chinese hamster ovary cells.
复制标题
靶向载体结构和基因转移方法影响中国仓鼠卵巢细胞APRT基因的靶向校正。
DOI：
10.1007/bf01232748
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发表时间：
1993
期刊：
影响因子：
--
通讯作者：
Seidman,MM
中科院分区：
文献类型：
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作者：
Nairn,RS;Adair,GM;Porter,T;Pennington,SL;Smith,DG;Wilson,JH;Seidman,MM
用含有仓鼠APRT序列的质粒转染中国仓鼠卵巢细胞（CHO），纠正了APRT基因第三外显子21个碱基的缺失。用含有3.2 kb APRT序列同源性的载体获得了0.3-3.0×10− 6范围内的靶向校正频率。为了检查载体构型对靶向基因校正的影响，在通过磷酸钙-DNA共沉淀或电穿孔转染之前，在载体中的两个位置之一处引入双链断裂。在载体中所含的同源区中的双链断裂产生插入型载体，而将断裂置于同源区之外产生复制型载体。两种线性载体构型的基因靶向产生靶向重组体与非靶向载体整合体的相等比例;然而，两种不同载体构型的靶向导致靶向重组产物的不同分布。通过Southern杂交分析66个独立的APRT+重组克隆表明，与整合载体配置相比，在双链型配置中用载体靶向产生更少的靶向整合体和更多的靶基因转化体。有针对性的重组与电穿孔比磷酸钙-DNA共沉淀的效率高约5倍，然而，这两种基因转移方法产生的有针对性的重组体的分布相似，这仅取决于靶向载体的配置。我们的研究结果表明，插入型和重复型基因打靶载体在基因校正实验中产生相似的总体打靶频率，但载体配置可以显着影响特定重组类型的产量。
A 21-bp deletion in the third exon of theAPRTgene in Chinese hamster ovary (CHO) cells was corrected by transfection with a plasmid containing hamsterAPRTsequences. Targeted correction frequencies in the range of 0.3–3.0×10−6were obtained with a vector containing 3.2 kb ofAPRTsequence homology. To examine the influence of vector configuration on targeted gene correction, a double-strand break was introduced at one of two positions in the vector prior to transfection by calcium phosphate-DNA coprecipitation or electroporation. A double-strand break in the region ofAPRThomology contained in the vector produced an insertion-type vector, while placement of the break just outside the region of homology produced a replacement-type vector. Gene targeting with both linear vector configurations yielded equivalent ratios of targeted recombinants to nontargeted vector integrants; however, targeting with the two different vector configurations resulted in different distributions of targeted recombination products. Analysis of 66 independent APRT+recombinant clones by Southern hybridization showed that targeting with the vector in a replacement-type configuration yielded fewer targeted integrants and more target gene convertants than did the integration vector configuration. Targeted recombination was about fivefold more efficient with electroporation than with calcium phosphate-DNA coprecipitation; however, both gene transfer methods produced similar distributions of targeted recombinants, which depended only on targeting vector configuration. Our results demonstrate that insertion-type and replacement-type gene targeting vectors produce similar overall targeting frequencies in gene correction experiments, but that vector configuration can significantly influence the yield of particular recombinant types.