Scheerer J B, Adair G M
University of Texas M. D. Anderson Cancer Center, Science Park-Research Division, Smithville 78957.
Mol Cell Biol. 1994 Oct;14(10):6663-73. doi: 10.1128/mcb.14.10.6663-6673.1994.
Using simple linear fragments of the Chinese hamster adenine phosphoribosyltransferase (APRT) gene as targeting vectors, we have investigated the homology dependence of targeted recombination at the endogenous APRT locus in Chinese hamster ovary (CHO) cells. We have examined the effects of varying either the overall length of targeting sequence homology or the length of 5' or 3' flanking homology on both the frequency of targeted homologous recombination and the types of recombination events that are obtained. We find an exponential (logarithmic) relationship between length of APRT targeting homology and the frequency of targeted recombination at the CHO APRT locus, with the frequency of targeted recombination dependent upon both the overall length of targeting homology and the length of homology flanking each side of the target gene deletion. Although most of the APRT+ recombinants analyzed reflect simple targeted replacement or conversion of the target gene deletion, a significant fraction appear to have arisen by target gene-templated extension and correction of the targeting fragment sequences. APRT fragments with limited targeting homology flanking one side of the target gene deletion yield proportionately fewer target gene conversion events and proportionately more templated extension and vector correction events than do fragments with more substantial flanking homology.
我们使用中国仓鼠腺嘌呤磷酸核糖转移酶（APRT）基因的简单线性片段作为靶向载体，研究了中国仓鼠卵巢（CHO）细胞内源性APRT基因座靶向重组的同源性依赖性。我们研究了改变靶向序列同源性的总长度或5'或3'侧翼同源性的长度对靶向同源重组频率以及所获得的重组事件类型的影响。我们发现，APRT靶向同源性的长度与CHO APRT基因座处靶向重组的频率之间存在指数（对数）关系，靶向重组的频率取决于靶向同源性的总长度以及靶基因缺失两侧同源性的长度。尽管分析的大多数APRT+重组体反映了靶基因缺失的简单靶向替换或转换，但相当一部分似乎是由靶基因模板化的靶向片段序列延伸和校正产生的。与具有更大量侧翼同源性的片段相比，在靶基因缺失一侧具有有限靶向同源性的APRT片段产生的靶基因转换事件成比例地更少，而模板化延伸和载体校正事件成比例地更多。
