Development and validation of a reporter cell line for rapid AAV quality control assessment

MethodsX

Roland S. Hilgarth¹  Huira C. Kopera²  Thomas M. Lanigan³  Tonya L. Kopas³ 

[1] Vector Core, Biomedical Research Core Facilities, University of Michigan, Ann Arbor, MI 48109, United States;Department of Human Genetics, University of Michigan, Ann Arbor, MI 48109, United States;Vector Core, Biomedical Research Core Facilities, University of Michigan, Ann Arbor, MI 48109, United States;

关键词: Viral vectors;    Fluorescence;    Transduction;   

DOI  :  

来源: DOAJ

【 摘 要 】

Adenovirus-associated virus is a powerful vector system for transducing cells in vivo. It is widely used in animal systems due to high transduction efficiency of non-dividing cells with more than a dozen serotypes that have preferential tissue tropism. The viral genome remains episomal in the nucleus but maintains sustained expression in terminally differentiated cells for several weeks to months. Despite the popularity of recombinant AAV (rAAV) vectors, quality control testing of the virus after production is largely limited to physical characteristics such as viral genomes/ml determinations and silver staining acrylamide gels to determine purity. Functional testing, in vivo, is not practical due to high cost and restricted access of animal care and long duration of the assay (2–3 weeks). Some functional testing can be accomplished in cultured cells such as HEK293 cells, but HEK293 cells limit the types of rAAV constructs that can be tested. Many rAAV constructs are designed to study neurons in the brain with neural-specific promoters and many are floxed with loxp sites to be “activated” only in Cre-expressing neurons in transgenic animals. To develop a reporter cell line for rapid rAAV quality control assessment of these neural-specific, floxed rAAV constructs, we used the lentiviral system to stably express Cre recombinase in the SH-SY5Y neuroblastoma cell line. • A simple and economic method to evaluate recombinant AAV in vitro. • Allows functional validation of rAAV across a wide range of serotypes and promoters. • Allows functional validation of Cre-dependent rAAV constructs.

【 授权许可】

Unknown