title: Efficient directional cloning of recombinant adenovirus vectors using DNA-protein complex.
abstract: We describe an efficient cloning system utilizing adenoviral DNA-protein complexes which allows the directional cloning of genes into adenoviral expression vectors in a single step. DNA-protein complexes derived from a recombinant adenovirus (AVC2.null) were isolated by sequential use of CsCl step gradients followed by isopycnic centrifugation in a mixture of CsCl and guanidine HCl. AVC2.null is an adenoviral expression vector containing unique restriction sites between the human CMV-IE promoter and the SV40 intron/polyadenylation site. Transgenes were prepared for cloning into this vector by introduction of compatible restriction sites by PCR. A vector expressing rat granulocyte-macrophage colony-stimulating factor (GM-CSF) was constructed using DNA-protein complex as well as by traditional recombination techniques. The efficacy of our adenoviral cloning system utilizing DNA-protein complex was two logs higher than that seen using homologous recombination. All viruses generated by directional ligation of the insert into the vector DNA-protein complexes contained the desired transgene in the correct orientation. This technique greatly simplifies and accelerates the generation of recombinant adenoviral vectors.
authors.affiliation: Clinical Gene Therapy Branch, National Human Genome Research Institute, National Institute of Health, Bethesda, MD 20892-1851, USA.
authors.forename: T
authors.initials: T
authors.lastname: Okada
authors.forename: W J
authors.initials: WJ
authors.lastname: Ramsey
authors.forename: J
authors.initials: J
authors.lastname: Munir
authors.forename: O
authors.initials: O
authors.lastname: Wildner
authors.forename: R M
authors.initials: RM
authors.lastname: Blaese
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D002586:Cesium; D002712:Chlorides; D003001:Cloning, Molecular / Q000379:methods*; D003587:Cytomegalovirus / Q000235:genetics; D017931:DNA Primers; D018076:DNA, Complementary; D004279:DNA, Viral / Q000302:isolation & purification; D015723:Gene Library; D005822:Genetic Vectors; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000096:biosynthesis*; D019791:Guanidine; D006801:Humans; D008264:Macrophages / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods; D051381:Rats; D011995:Recombination, Genetic; D013539:Simian virus 40 / Q000235:genetics; D014162:Transfection / Q000379:methods
chemical_list: D002712:Chlorides; D017931:DNA Primers; D018076:DNA, Complementary; D004279:DNA, Viral; D002586:Cesium; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; C028019:cesium chloride; D019791:Guanidine