Selection of rare event cells expressing [beta]-galactosidase

Abstract

The ACAS Interactive Laser Cytometer was used to screen and select mammalian cells in culture infected with a retrovirus construct containing the Escherichia coli lacZ and neomycin resistance genes. The product of expression of the lacZ gene, [beta]-galactosidase, can be quantitated using the fluorescent substrate fluorescein di-[beta]-d-galactopyranoside (FDG). NIH 3T3 cells infected with the retrovirus construct were selected for neomycin resistance and individual colonies were cloned. These cells served as a positive control, and uninfected parent NIH 3T3 cells were used as the negative control. Autofluorescence and background fluorescence using FDG were examined on uninfected NIH 3T3 cells and found to be negligible. The fluorescence assay was performed on individual adherent cells in culture. Our results indicate that cells expressing the recombinant retrovirus product, [beta]-galactosidase, can be viably stained in culture, analyzed, sorted, and cloned without the cells being trypsinized or removed from their natural growth surface. The assay is sensitive enough to detect lacZ+ and lacZ- cells and also capable of discriminating between high and low expressers of [beta]-galactosidase. This technology could be a powerful tool in gene therapy and gene regulation studies. By using the [beta]-galactosidase gene as the reporter molecule in the constructs containing the gene of interest, selection of infected/transfected cells, infection/transfection efficiency, promoter strength, and specificity can be measured with minimal manipulation soon after the infection/transfection process. This technique can be further extended for sorting mutant cells, hybridomas, and rare event occurrences.