Gossen J A, de Leeuw W J, Molijn A C, Vijg J
Ingeny B.V., The Netherlands.
Biotechniques. 1993 Apr;14(4):624-9.
A method for the efficient rescue of lac operator containing plasmids from transgenic mouse genomic DNA is described. The method is based on the high affinity of the LacI repressor protein for the lac operator sequence. Using the LacI repressor protein conjugated to magnetic beads, more than 95% of plasmid sequences could be purified from restriction enzyme digested genomic DNA. After circularization, the plasmids were introduced into Escherichia coli by means of electroporation. Since the plasmid was cloned into a bacteriophage lambda vector, the efficiency of plasmid rescue could easily be compared with in vitro packaging. Our results indicate that plasmid rescue is about 25 times more efficient. Application of this method should be especially useful with transgenic mouse models harboring LacZ plasmid shuttle vectors for studying spontaneous or induced mutations in vivo.
本文描述了一种从转基因小鼠基因组DNA中高效拯救含乳糖操纵子质粒的方法。该方法基于乳糖抑制蛋白（LacI）对乳糖操纵子序列的高亲和力。使用与磁珠偶联的LacI抑制蛋白，超过95%的质粒序列可从经限制性内切酶消化的基因组DNA中纯化出来。环化后，通过电穿孔将质粒导入大肠杆菌。由于该质粒被克隆到噬菌体λ载体中，因此质粒拯救效率可轻松与体外包装进行比较。我们的结果表明，质粒拯救效率约高25倍。该方法对于携带LacZ质粒穿梭载体的转基因小鼠模型在体内研究自发或诱导突变尤其有用。
