Construction of a PAC vector system for the propagation of genomic DNA in bacterial and mammalian cells and subsequent generation of nested deletionsin individual library members

The BAC and PAC cloning systems allow investigators to propagate large geno mic DNA fragments up to 300 kb in size in E. colicells,We describe a new PA C shuttle vector that can be propagated in both bacterial and human cells. Specifically, the fl cloning vector pAd10sacBII was modified by the inserti on of a puromycin-resistance gene (pnc), the Epstein-Barr Virus (EBV) laten t replication origin oriP,and the EBV EBNA1 gene. Transfection studies in H EK 293 cells demonstrated that the modified vector was stably maintained as an episome for at least 30 generations. And since pJCPAC-Mam1 contains a l oxP site, genomic DNA cloned into this vector can be subjected to loxP-Cre- mediated deletion events. The transposon vector pTnPGKpuro/loxP was modifie d to make this system amenable to propagation in human cells by inserting p nc, oriP, and EBNA1 elements into the vector (Chatterjee, P.K., Coren, J.C. , 1997, Isolating large nested deletions in PACs and BACs by in vivo select ion of P1 headful-packaged products of Cre-catalyzed recombination between the loxP site in PAC and BAC and one introduced in transposition. NAR 25, 2 205-2212.). pTnPGKpuro/loxP-EBV was then used to generate deletions in an i ndividual library member to demonstrate that all of the deletions still con tain the required eukaryotic elements and that they were nested. All librar y members constructed in pJCPAC-Mam1 can be directly transformed into human cells to assess function. And the deletion technology can be used to aid i n delineating the boundaries of genes and other cis-acting elements, (C) 20 01 Elsevier Science B.V. All rights reserved.