tailieunhanh - Báo cáo khoa học: "A simplified in vitro ligation approach to clone an E1B55k-deleted double-targeted conditionally-replicative adenovirus Yosef S Haviv"

Tuyển tập báo cáo các nghiên cứu khoa học quốc tế ngành y học dành cho các bạn tham khảo đề tài: A simplified in vitro ligation approach to clone an E1B55k-deleted double-targeted conditionally-replicative adenovirus Yosef S Haviv | Virology Journal BioMed Central Open Access Methodology A simplified in vitro ligation approach to clone an E B55k-deleted double-targeted conditionally-replicative adenovirus Yosef S Haviv Address Department of Medicine Hadassah-Hebrew University Medical Center Jerusalem 91120 Israel Email Yosef S Haviv - yhaviv@ Published 7 February 2009 Received 26 January 2009 Accepted 7 February 2009 Virology journal 2009 6 18 doi 1743-422X-6-18 This article is available from http content 6 1 18 2009 Haviv licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License http licenses by which permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited. Abstract Background Construction of conditionally-replicative Adenovirus CRAd is complex and timeconsuming. 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 E l 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 doubletargeted El B55k-deleted CRAd. The method allowed the incorporation of a tumor-specific promoter . the heat-shock protein 70 hsp70 promoter upstream of Ela deletion of the EIB55kgene and HR-free cloning of the recombined El 55kgene into the Ad genome. The genetic structure of the CRAd was confirmed using restriction analysis and PCR. The replication rate of the hsp70El A55k CRAd was l .5-2 of Ad without E l A55k deletion. Conclusion A 3-step cloning approach can generate a double-targeted ElB55k-deleted .