TY - JOUR
T1 - A simplified in vitro ligation approach to clone an E1B55k-deleted double-targeted conditionally-replicative adenovirus
AU - Haviv, Yosef S.
N1 - Funding Information:
This study was performed during a research fellowship at the Division of Human Gene Therapy, University of Alabama at Birmingham, Birmingham, AL, USA and was funded by the by the Birmingham Jewish Federation. The author wishes to thank David T. Curiel, Jerry L. Blackwell, Yasuo Adachi, Hongju Wu and Koichi Takayama for their support, helpful discussions and valuable reagents. Ming Wang performed the real-time PCR. Hui Li is acknowledged for technical support. Current research is funded by the German-Israeli Foundation grant no. 817/2004, Israel Science Foundation grant no. 573/03, the Israeli Ministry of Health, Chief Scientist Office, and the Hadassah Women's health fund.
PY - 2009/3/9
Y1 - 2009/3/9
N2 - Background: Construction of conditionally-replicative Adenovirus (CRAd) is complex and time-consuming. While homologous recombination (HR) using a two-plasmid system in bacteria is commonly used to generate CRAds, alternative methods may be required when HR fails. Previously, in vitro ligation has been suggested to facilitate construction of E1/E3-deleted, replication-incompetent Ad vectors. However, in vitro ligation has only rarely been used to generate CRAds and may be a complex procedure for molecular biologists who are not experts in the field. Methods and Results: A modified in vitro ligation approach was developed to construct a double-targeted, E1B55k-deleted CRAd. The method allowed the incorporation of a tumor-specific promoter, e.g. the heat-shock protein 70 (hsp70) promoter, upstream of E1a, deletion of the E1B55k gene, and HR-free cloning of the recombined E1δ55k gene into the Ad genome. The genetic structure of the CRAd was confirmed using restriction analysis and PCR. The replication rate of the hsp70E1δ55k CRAd was 1.5-2% of Ad without E1δ55k deletion. Conclusion: A 3-step cloning approach can generate a double-targeted, E1B55k-deleted CRAd using a straight-forward, modified in vitro ligation procedure.
AB - Background: Construction of conditionally-replicative Adenovirus (CRAd) is complex and time-consuming. While homologous recombination (HR) using a two-plasmid system in bacteria is commonly used to generate CRAds, alternative methods may be required when HR fails. Previously, in vitro ligation has been suggested to facilitate construction of E1/E3-deleted, replication-incompetent Ad vectors. However, in vitro ligation has only rarely been used to generate CRAds and may be a complex procedure for molecular biologists who are not experts in the field. Methods and Results: A modified in vitro ligation approach was developed to construct a double-targeted, E1B55k-deleted CRAd. The method allowed the incorporation of a tumor-specific promoter, e.g. the heat-shock protein 70 (hsp70) promoter, upstream of E1a, deletion of the E1B55k gene, and HR-free cloning of the recombined E1δ55k gene into the Ad genome. The genetic structure of the CRAd was confirmed using restriction analysis and PCR. The replication rate of the hsp70E1δ55k CRAd was 1.5-2% of Ad without E1δ55k deletion. Conclusion: A 3-step cloning approach can generate a double-targeted, E1B55k-deleted CRAd using a straight-forward, modified in vitro ligation procedure.
UR - http://www.scopus.com/inward/record.url?scp=61449208225&partnerID=8YFLogxK
U2 - 10.1186/1743-422X-6-18
DO - 10.1186/1743-422X-6-18
M3 - Article
C2 - 19200390
AN - SCOPUS:61449208225
SN - 1743-422X
VL - 6
JO - Virology Journal
JF - Virology Journal
M1 - 18
ER -