Determination of Functional Viral Titer by Drug-Resistance Colony Assay, Expression of Green Fluorescent Protein, and -Galactoside Staining

With this method, antibiotic selection of the infected cells gives rise to a countable number of colonies after 7–10 d (1 –4 ). Other reporter genes are based on green fluorescent protein (GFP; ref. 5 ) or the Escherichia coli lacZ gene, which encodes for β-galactosidase for X-gal staining (4 ,6 ). GFP-expressing cells can be easily visualized under an ultraviolet microscope. The main advantage is that GFP expression can be detected in living cells (5 ). Retroviral and lentiviral transfer vectors can be engineered with an internal phosphoglycerate kinase (pgk) promoter driving the expression of a bacterial gene that confers resistance to a certain antibiotic (1 , 2 ). Usually, the bacterial genes used are based on neomycin phosphotransferase, hygromycin B phosphotransferase, or puromycin N -acetyltransferase, which render mammalian cells resistant to G418, hygromycin B, and puromycin, respectively (1 ). In addition, either the internal pgk or SV40 early promoter can drive the expression of lacZ gene (4 ,6 ) or GFP (5 ). Functional titer does not provide a consistent measurement of virion concentration because it depends upon the transduction efficiency of the cell line being used to determine the titer. Therefore, several investigators use preferentially direct quantification for determining virus particle concentration. There are several bibliographic references for the counting of selected colonies (3 ,4 ).