Fisher P B, Babiss L E, Weinstein I B, Ginsberg H S
Proc Natl Acad Sci U S A. 1982 Jun;79(11):3527-31. doi: 10.1073/pnas.79.11.3527.
A recently isolated cloned cell line of Fischer rat embryo fibroblasts (CREF) can be transformed at high frequency by adenovirus serotype 5 (Ad5). The CREF cells display a near diploid karyotype, do not crisscross at confluency, can be maintained at confluency for greater than 7 wk at 36 degrees C, and do not form macroscopic colonies when seeded in agar. Transformed cells are identified by their altered morphology and the presence of adenovirus DNA sequences in the transformants, which can be demonstrated by lysing cells directly on nitrocellulose filters and hybridizing with 32P-labeled Ad5 DNA (spot hybridization). The frequency of transformation at 36 degrees C is approximately equal to 2 x 10(-4) with wild-type Ad5 and approximately equal to 2 x 10(-3) with the temperature-sensitive mutant H5ts125. Southern blot hybridization analysis indicates that five out of six wild-type Ad5- and six out of six H5ts125-transformed CREF clones isolated at 36 degrees C contain the complete integrated Ad5 genome. Preliminary characterization of four transformed clones (two wild-type and two H5ts125) indicates that, even though transformation was done in CREF cells (a clonal cell line), they differ in their biological properties, such as saturation density and anchorage dependence.
最近分离出的费希尔大鼠胚胎成纤维细胞（CREF）克隆细胞系可被5型腺病毒（Ad5）高频转化。CREF细胞呈现近二倍体核型，在汇合时不交叉，在36℃下可在汇合状态维持超过7周，接种于琼脂中时不形成肉眼可见的菌落。通过形态改变和转化体中腺病毒DNA序列的存在来鉴定转化细胞，这可通过直接在硝酸纤维素滤膜上裂解细胞并与32P标记的Ad5 DNA杂交（斑点杂交）来证明。在36℃下，野生型Ad5的转化频率约为2×10^(-4)，温度敏感突变体H5ts125的转化频率约为2×10^(-3)。Southern印迹杂交分析表明，在36℃下分离的6个野生型Ad5转化的CREF克隆中有5个以及6个H5ts125转化的CREF克隆中有6个含有完整整合的Ad5基因组。对4个转化克隆（2个野生型和2个H5ts125）的初步表征表明，尽管转化是在CREF细胞（一种克隆细胞系）中进行的，但它们在生物学特性上存在差异，如饱和密度和贴壁依赖性。
