2 LARGE INSERT VECTORS, LAMBDA-PS AND LAMBDA-KO, FACILITATE RAPID MAPPING AND TARGETED DISRUPTION OF MAMMALIAN GENES

The construction and the testing of two lambda phage vectors are descr ibed that greatly simplify the tasks of mapping genomic DNA and making replacement-type gene-targeting vectors for mammalian cells from a li brary of isogenic genomic DNA. The first vector lambda PS, accommodate s lip to 20 kb and allows inserts to be automatically subcloned in pla smid form because of the presence of loxP sites flanking the insert. T he second vector lambda KO, accommodates rep to 16.7 kb and allows ins erts to be automatically subcloned as plasmids containing HSVtk genes that are positioned flanking the inserted genomic DNA. We have prepare d highly redundant libraries from genomic DNA of 129/Sv-strain mice fo r the construction of targeting vectors. In our scheme, the locus of i nterest is characterized using a library made in lambda PS. For instan ce, suitable flanking probes can be derived to determine targeting eve nts. The final targeting construct with flanking HSVtk genes is obtain ed using the lambda KO cloning vector. The entire procedure is exempli fied by successful targeting of tile X-linked mouse hprt locus.