Bobovnikova Y, Kim S J, Wetmur J G
Department of Microbiology, Mount Sinai School of Medicine, New York, NY 10029, USA.
Gene. 1996 Apr 17;170(1):39-44. doi: 10.1016/0378-1119(95)00840-3.
A P1-based cloning system has been tested which depends upon (i) in vitro selection for vectors containing inserts mediated by methyltransferase (MTase) protection, and (ii) in vivo vector arm Cre-mediated recombination following electroporation. Specifically, chromosomal DNA was digested with BglII, dephosphorylated, methylated with BamHI MTase and ligated into the BamHI site of the vector, thereby destroying that site. Subsequent BamHI digestion acted as the in vitro selection, eliminating vector religation products prior to electroporation into cells expressing the Cre recombinase. Electroporation with linearized vector gave approx. 10(6) transformants per microgram vector, depending on vector concentration. Cloning of BglII fragments of gamma DNA using the in vitro selection system led to 1.3-4-fold fewer transformants per microgram vector. Plasmids recovered from these clones were all found to contain a gamma BglII fragment and the representation of fragments in the clones was independent of the length of the fragments. Both in vitro selection and electroporation are applicable to library construction using size-selected human DNA, with size selection either before or after ligation and BamHi digestion.
一种基于P1的克隆系统已得到测试，该系统依赖于：（i）通过甲基转移酶（MTase）保护介导的对含有插入片段的载体进行体外筛选，以及（ii）电穿孔后体内载体臂Cre介导的重组。具体而言，染色体DNA用BglII消化、去磷酸化，用BamHI MTase甲基化，然后连接到载体的BamHI位点，从而破坏该位点。随后的BamHI消化作为体外筛选，在电穿孔导入表达Cre重组酶的细胞之前消除载体自连产物。根据载体浓度，用线性化载体进行电穿孔，每微克载体可产生约10⁶个转化体。使用体外筛选系统克隆γ DNA的BglII片段，每微克载体产生的转化体数量减少了1.3至4倍。从这些克隆中回收的质粒均发现含有γ BglII片段，并且克隆中片段的代表性与片段长度无关。体外筛选和电穿孔都适用于使用大小选择的人类DNA构建文库，大小选择可在连接和BamHI消化之前或之后进行。
