Hamada Y, Tsujimoto Y, Ishiura M, Suzuki Y
Gene. 1983 Oct;24(2-3):245-53. doi: 10.1016/0378-1119(83)90085-9.
Recombinant Charon 4A phages accommodating the Herpes simplex virus (HSV-1) thymidine kinase (tk) gene, the ampicillin-resistance (ApR) gene, and the replication origin of pBR322 were constructed. The phage DNA was introduced into mouse Ltk- cells by a free DNA transfer method or phage-mediated DNA transfer method [Ishiura et al., Mol. Cell. Biol. 2 (1982) 607]. Analyses of the physical state of the transferred DNA in the recipient cell genome showed that a DNA fragment as long as 12.7 kb was integrated intact into 67% and less than 40% of the Ltk- transformant cells by phage-mediated DNA transfer and by free DNA transfer, respectively. We also developed a new rapid method for recovery of the transferred gene from the Ltk+ cell into Escherichia coli; the method depends on the fact that the recombinant lambda phage carrying the ApR gene and replication origin of pBR322 transduces lambda-lysogenic bacteria to ApR and is maintained as a plasmid. Using this method the HSV-1 tk gene from one Ltk+ transformant was rapidly and successfully recovered without any rearrangement of the target sequence.
构建了携带单纯疱疹病毒1型（HSV-1）胸苷激酶（tk）基因、氨苄青霉素抗性（ApR）基因和pBR322复制起点的重组Charon 4A噬菌体。通过游离DNA转移法或噬菌体介导的DNA转移法将噬菌体DNA导入小鼠Ltk-细胞[石浦等人，《分子与细胞生物学》2（1982）607]。对受体细胞基因组中转移DNA的物理状态分析表明，通过噬菌体介导的DNA转移和游离DNA转移，分别有67%和不到40%的Ltk-转化细胞完整整合了长达12.7 kb的DNA片段。我们还开发了一种从Ltk+细胞中快速回收转移基因到大肠杆菌中的新方法；该方法基于携带ApR基因和pBR322复制起点的重组λ噬菌体将λ溶原性细菌转导为ApR并作为质粒维持这一事实。使用该方法，从一个Ltk+转化体中快速且成功地回收了HSV-1 tk基因，且目标序列没有任何重排。
