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Flow Cytometric Application of Helper Adenovirus (HAd) Containing GFP Gene Flanked by Two Parallel lox P Sites to Evaluation of 293 Cre‐Complementing Cell Line and Monitoring of HAd in Gutless Ad Production
Author(s) -
Park Min Tae,
Hwang SuJeong,
Lee Gyun Min
Publication year - 2004
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp034248e
Subject(s) - green fluorescent protein , flow cytometry , biology , microbiology and biotechnology , gene , western blot , genetics
Gutless adenoviruses (GAds), namely, all gene‐deleted adenoviruses, were developed to minimize their immune responses and toxic effects for a successful gene delivery tool in gene therapy. The Cre/ lox P system has been widely used for GAd production. To produce GAd with a low amount of helper adenovirus (HAd) as byproduct, it is indispensable to use 293Cre cells expressing a high level of Cre for GAd production. In this study, we constructed the HAd containing enhanced green fluorescent protein gene flanked by two parallel lox P sites (HAd/GFP). The use of HAd/GFP with flow cytometry allows one to select 293Cre cells expressing a high level of Cre without using conventional Western blot analysis. Unlike conventional HAd titration methods such as plaque assay and end‐point dilution assay, it also allows one to monitor rapidly the HAd as byproduct in earlier stages of GAd amplification. Taken together, the use of HAd/GFP with flow cytometry facilitates bioprocess development for efficient GAd production.