Efficient generation of recombinant adenoviruses using adenovirus DNA-terminal protein complex and a cosmid bearing the full-length virus genome.

Abstract

An efficient method of constructing recombinant adenoviruses (Ads)

Regulatable production of insulin from primary-cultured hepatocytes: insulin production is up-regulated by glucagon and cAMP and down-regulated by insulin

“…36 The presence of terminal protein protects the Ad5-dlX DNA from exonuclease attack, therefore a homologous recombination between the pAdex1cw and Ad5-dlX becomes far more efficient in 293 cells. After selecting recombinant adenoviruses with an insulin expression vector, AdP 3 ␤Ins and AdP 0 ␤Ins ( Figure 1) were propagated for use in insulin gene transfer to hepatocytes.…”

“…15 This insulin expression unit was cut from the plasmid and inserted into the SwaI site of the cassette cosmid pAdex1cw (pAdP 3 ␤Ins and pAdP 0 ␤Ins). 36 Recombinant virus was generated by a homologous recombination between either pAdP 3 ␤Ins or pAdP 0 ␤Ins and the EcoT22I-digested DNA-terminal protein complex of Ad5-d1X 36 in human embryonic kidney 293 cells (ATCC, CRL 1573). Because the 293 cells are integrated with the adenoviral E1A region, E1A-deleted adenoviral recombinant DNA can propagate in the cells.…”

“…Thus, we decided to construct a regulatable insulin expression system by using an adenoviral vector. 36,37 Using a recombinant adenoviral vector, we observed the production of mature insulin by primary cultured rat hepatocytes and found that insulin secretion from hepatocytes is up-regulated by glucagon and dibutyryl cAMP (dbcAMP) and down-regulated by insulin itself. Insulin production was augmented by the presence of wortmannin, thought to block the insulin receptor-mediated signaling pathway, possibly by inhibiting phosphatidylinositol (PI)-3-kinase.…”

Dominant negative c-Jun gene transfer inhibits vascular smooth muscle cell proliferation and neointimal hyperplasia in rats

“…26 TAM67 has the DNA-binding domain of wild c-Jun. Recombinant replication-defective E1-and E3-adenoviral vectors expressing TAM67 gene (Ad-DNc-Jun) were constructed using adenovirus expression vector kit (TAKARA Biomedicals, Japan), according to the Gene Therapy method of Miyake et al 27 cDNA encoding TAM67 was placed into a cassette cosmid vector PaxCAwt which possesses CAG promoter comprising a cytomegalovirus enhancer, chicken ␤-actin promoter and rabbit ␤-globin poly A signal. A recombinant adenovirus was constructed by in vitro homologus recombination in 293 cells using the above cosmid vector PaxCAwt containing TAM67 cDNA and the adenovirus DNA-terminal protein complex.…”

A protocol for rapid generation of recombinant adenoviruses using the AdEasy system

“…However, this proved to be the rate-limiting step in the production of recombinant adenoviruses owing to the inefficient and somewhat unpredictable nature of homologous recombination in mammalian cells. Yeast and bacterial systems have, therefore, been explored for this purpose 11,12,[14][15][16][17][18][19][20] . Once a DNA molecule incorporating both the backbone and shuttle vector sequences has been generated in such microbial systems, the cloned DNA can be transfected into mammalian packaging lines for virus production.…”