Conrad S E, Liu C P, Botchan M R
Science. 1982 Dec 17;218(4578):1223-5. doi: 10.1126/science.6293055.
A 311-base pair fragment containing the SV40 origin of replication was linked to the chicken thymidine kinase gene on a recombinant plasmid. This molecule was transfected into human 143 thymidine kinase-deficient (TK-) cells, and colonies positive for thymidine kinase were selected. When cell lines derived from these colonies were fused to permissive simian cells that produce SV40 T antigen, the recombinant plasmid excised itself from the human cellular genome and replicated with a high copy number per cell. These results show that this segment of the viral genome is the only sequence required in cis to mediate SV40 excision and replication upon fusion to permissive cells. In addition, we have shown that excised plasmids apparently identical to the input DNA can be efficiently rescued in Escherichia coli. SV40 excision and replication may therefore be useful for the recovery of cloned genes from eukaryotic cells.
一个包含SV40复制起点的311个碱基对的片段与重组质粒上的鸡胸苷激酶基因相连。该分子被转染到人143胸苷激酶缺陷（TK-）细胞中，并筛选出胸苷激酶阳性的菌落。当源自这些菌落的细胞系与产生SV40 T抗原的允许性猴细胞融合时，重组质粒从人类细胞基因组中自行切除，并在每个细胞中以高拷贝数复制。这些结果表明，病毒基因组的这一片段是在与允许性细胞融合时介导SV40切除和复制所需的唯一顺式作用序列。此外，我们已经表明，与输入DNA明显相同的切除质粒可以在大肠杆菌中有效地拯救出来。因此，SV40切除和复制可能有助于从真核细胞中回收克隆基因。
