ISOLATING LARGE NESTED DELETIONS IN BACTERIAL AND P1 ARTIFICIAL CHROMOSOMES BY IN-VIVO P1 PACKAGING OF PRODUCTS OF CRE-CATALYZED RECOMBINATION BETWEEN THE ENDOGENOUS AND A TRANSPOSED LOXP SITE

A general approach for isolating large nested deletions in P1 artifici al chromosomes (PACs)and bacterial artificial chromosomes (BACs) by re trofitting with a loxP site-containing Tn10 mini-transposon is describ ed. Cre-mediated recombination between the loxP site existing in these clones and one introduced by transposition leads to deletions and inv ersions of the DNA between these sites. Large deletions are selectivel y recovered by transducing the retrofitted PAC or BAC clones with P1 p hage. The requirement that both loxP sites in the cointegrate be packa ged into a P1 head ensures that only large deletions are rescued. PCR analyses identified these deletions as products of legitimate recombin ation between loxP sites mediated by Cre protein. BACs produce deletio ns much more efficiently than PACs although the former cannot be induc ed to greater than unit copy in cells. Mammalian cell-responsive antib iotic resistance markers are introduced as part of the transposon into genomic clone deletions for subsequent functional analysis. Most impo rtantly, the loxP site retrofitting and P1 transduction can be perform ed in the same bacterial host containing these clones directly isolate d from PAC or BAC libraries. These procedures should facilitate physic al and functional mapping of genes and regulatory elements in these la rge plasmids.