Nehls M, Messerle M, Sirulnik A, Smith A J, Boehm T
University of Freiburg, FRG.
Biotechniques. 1994 Oct;17(4):770-5.
The construction and the testing of two lambda phage vectors are described that greatly simplify the tasks of mapping genomic DNA and making replacement-type gene-targeting vectors for mammalian cells from a library of isogenic genomic DNA. The first vector, lambda PS, accommodates up to 20 kb and allows inserts to be automatically subcloned in plasmid form because of the presence of loxP sites flanking the insert. The second vector, lambda KO, accommodates up to 16.7 kb and allows inserts to be automatically subcloned as plasmids containing HSVtk genes that are positioned flanking the inserted genomic DNA. We have prepared highly redundant libraries from genomic DNA of 129/Sv-strain mice for the construction of targeting vectors. In our scheme, the locus of interest is characterized using a library made in lambda PS. For instance, suitable flanking probes can be derived to determine targeting events. The final targeting construct with flanking HSVtk genes is obtained using the lambda KO cloning vector. The entire procedure is exemplified by successful targeting of the X-linked mouse hprt locus.
本文描述了两种λ噬菌体载体的构建和测试方法，这些方法极大地简化了从同基因基因组DNA文库中绘制基因组DNA图谱以及制备用于哺乳动物细胞的置换型基因靶向载体的任务。第一种载体λPS，可容纳高达20 kb的片段，由于插入片段两侧存在loxP位点，可使插入片段自动以质粒形式亚克隆。第二种载体λKO，可容纳高达16.7 kb的片段，可使插入片段自动亚克隆为含有位于插入的基因组DNA两侧的HSVtk基因的质粒。我们已经从129/Sv品系小鼠的基因组DNA制备了高度冗余的文库用于构建靶向载体。在我们的方案中，使用在λPS中构建的文库对感兴趣的基因座进行表征。例如，可以获得合适的侧翼探针来确定靶向事件。使用λKO克隆载体获得带有侧翼HSVtk基因的最终靶向构建体。通过成功靶向X连锁的小鼠hprt基因座对整个过程进行了举例说明。
