Efficient dual transcomplementation of adenovirus E1 and E4 regions from a 293-derived cell line expressing a minimal E4 functional unit.

摘要: Transgene expression after the administration of recombinant adenovirus with E1 deleted is constantly transient. It admitted that E1A-substituting activities cellular or viral origin allow antigen synthesis and trigger cytotoxic lymphocyte-mediated clearance recipient cells. Our approach to solving this problem relies on additional deletion E4 region from vector backbone as upregulates gene at both transcriptional posttranscriptional levels. As a prerequisite construction doubly defective adenoviruses, we investigated possibility transcomplementing functions within single cell. In particular, distal ORF6+ORF7 segment locus type 5 was cloned under control dexamethasone-inducible mouse mammary tumor virus long terminal repeat. Following transfection into 293 cells, clone IGRP2 retained characterized it can rescue growth defect all E1+ E4- adenoviral deletants tested. DNA RNA analysis experiments verified promoter drives unit permits its bona fide alternative splicing, generating ORF6/7 mRNA in addition ORF6-expressing primary transcript. Importantly, cells sustain cell confluence for period longer than parental plaque purification E1- viruses. The dual regulatory genes demonstrated by construction, purification, helper-free propagation lacZ-encoding adenoviruses harboring different deletions. addition, emergence, if any, replicative particles during novel packaging line will be drastically impaired only limited has been integrated.