Journal Article

Efficient directional cloning of recombinant adenovirus vectors using DNA-protein complex

Takashi Okada, W. Jay Ramsey, Jamalah Munir, Oliver Wildner and R. Michael Blaese

in Nucleic Acids Research

Volume 26, issue 8, pages 1947-1950
Published in print April 1998 | ISSN: 0305-1048
Published online April 1998 | e-ISSN: 1362-4962 | DOI: https://dx.doi.org/10.1093/nar/26.8.1947
Efficient directional cloning of recombinant adenovirus vectors using DNA-protein complex

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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.

Journal Article.  2087 words.  Illustrated.

Subjects: Chemistry ; Biochemistry ; Bioinformatics and Computational Biology ; Genetics and Genomics ; Molecular and Cell Biology

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