Flow cytometric application of helper adenovirus (HAd) containing GFP gene flanked by two parallel loxP sites to evaluation of 293 cre-complementing cell line and monitoring of HAd in Gutless Ad production.
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/loxP 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 loxP 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.
Park
Min Tae
MT
Department of Biological Sciences, Korea Advanced Institute of Science and Technology 373-1, Kusong-Dong, Yusong-Gu, Daejon 305-701, Korea.
Hwang
Su-Jeong
SJ
Lee
Gyun Min
GM
D000256:Adenoviridae / Q000235:genetics* / Q000302:isolation & purification* / Q000502:physiology; D002460:Cell Line; D002469:Cell Separation / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods*; D017930:Genes, Reporter / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D019426:Integrases / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D007668:Kidney / Q000821:virology*; D014764:Viral Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D014776:Virus Cultivation / Q000379:methods*
D014764:Viral Proteins; D049452:Green Fluorescent Proteins; C045073:Cre recombinase; D019426:Integrases