title: Rapid titration of retroviral vectors using a β-lactamase protein fragment complementation assay.
abstract: The availability of rapid and quantitative titration assays for retroviral vectors is important, especially in the context of clinical applications. In this report, we describe a novel assay to titrate lentiviral and gamma retroviral vectors. This rapid assay is based on protein fragment complementation involving the N-terminal (Bla1) and the C-terminal (Bla2) fragments of TEM-1 β-lactamase (BLAK). The Bla1 protein fragment is incorporated in the vector's envelope during vector production. Bla1-bearing vectors are titrated on Bla2-expressing cells. Upon transduction, Bla1 and Bla2 heterodimerize and restore BLAK's enzymatic function. The enzymatic activity of BLAK is quantified by flow cytometry using the green fluorescent CCF2/AM substrate, which is converted into a blue fluorescent product. The enzymatic conversion of the CCF2/AM substrate was found to be directly related to vector entry, as a neutralizing antibody completely blocked the conversion. The titers obtained using this rapid assay correlated well with the titers measured by functional transduction assays. The whole assay can be finished within 8 h. Thus, it is considerably less time consuming compared with other transduction-based titration assays for lentiviral and gamma retroviral vectors.
authors.affiliation: Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, FDA, Bethesda, MA, USA.
authors.forename: W
authors.initials: W
authors.lastname: Ou
authors.forename: M P
authors.initials: MP
authors.lastname: Marino
authors.forename: C
authors.initials: C
authors.lastname: Lu
authors.forename: J
authors.initials: J
authors.lastname: Reiser
mesh_terms: D017988:Gammaretrovirus / Q000235:genetics*; D018014:Gene Transfer Techniques; D005816:Genetic Complementation Test; D005822:Genetic Vectors / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D001618:beta-Lactamases / Q000235:genetics*
chemical_list: D001618:beta-Lactamases