An efficient and flexible system for construction of adenovirus vectors with insertions or deletions in early regions 1 and 3.

Abstract

Human adenoviruses (Ads) are attracting considerable attention because oftheir potential utility for gene transfer and gene therapy, for development of live viral vectored vaccines, and for protein expression in mammalian cells. Engineering Ad vectors for these applications requires a variety of reagents in the form of Ads and bacterial plasmids containing viral DNA sequences and requires different strategies for construction of vectors for different purposes. To simplify Ad vector construction and develop a procedure with maximum flexibility, efficiency, and cloning capacity, we have developed a vector system based on use of AdS DNA sequences cloned in bacterial plasmids. Expanded deletions in early region 1 (3180 bp) and early region 3 (2690 or 3132 bp) can be combined in a single vector that should have a capacity for inserts of up to 8.3 kb, enough to accommodate the majority of cDNAs encoding proteins with regulatory elements. Genes can be inserted into either early region 1 or 3 or both and mutations or deletions can be readily introduced elsewhere in the viral genome. To illustrate the flexibility of the system, we have introduced a wild-type early region 3 into the vectors, and to illustrate the high capacity for inserts, we have isolated a vector with two genes totaling 7.8 kb. (17). After 8-10 days, plaques were isolated and expanded, and viral DNA was analyzed by restriction enzyme digestion as described (12, 16). [35S]Methionine labeling, immunoprecipitation, and SDS/PAGE were carried out as described (16,18). Densitometry was performed using the LKB Ultroscan XL enhanced laser densitometer. Construction of adenovirus (Ad

E1A-induced apoptosis does not prevent replication of adenoviruses with deletion of E1b in majority of infected cancer cells

“…The pBHGE3 plasmid 34 contains Ad genomic DNA. The pBHGE3 has a partial deletion of the E1 region and the essential viral-packaging signal.…”

“…Plasmid pDE1spA1 is an Ad shuttle plasmid with an E1 deletion from bp 342 to 3523. 34 Both of these Ad plasmids were kindly provided by Dr F Graham.…”

Profiling of genes which are differentially expressed in mouse liver in response to adenoviral vectors and delivered genes

“…Recombination of the shuttle vector expressing these transgenes with the pbGH10 backbone (Microbix) was performed as described. 29 Scale up and purification of recombinant adenovirus The scale up and purification of the adenoviral constructs was performed as previously described. 9 Following recombination, each adenoviral construct was amplified in 911 cells 30 (Introgene, Leiden, The Netherlands) from a purified plaque which had been demonstrated to express the transgene.…”

Comparison of HSV-1 thymidine kinase-dependent and -independent inhibition of replication-competent adenoviral vectors by a panel of drugs

“…14,15 Briefly, beginning with a first-generation adenovirus early region 1 (E1)-and adenovirus early region 3 (E3)-deleted adenoviral vector, we generated the replication-competent adenoviral vector Ad.OW34, which harbors in the E1 region an HSV-tk-IRES 91,92 -Ad5 E1 expression cassette driven by the human cytomegalovirus immediate-early (CMV-IE) promoter flanked upstream by the Ad5 packaging sequence and downstream by the Ad5 pIX. Ad.OW34 was generated by homologous recombination of pAd.CMV-TK E1 and pBHG10 93 in 293 cells, as described previously. 94 The replication-competent adenoviral reporter gene vector Ad.OW94 was generated by substituting the HSV-tk gene of the Ad.OW34 vector with the cDNA encoding enhanced GFP from the plasmid pGreenLantern-1 (Life Technologies, Grand Island, NY).…”