Recovery of a rare clone from a population of unstable retroviral vector-expressing mammalian cells using a new RNA extraction and slot-blot protocol

Although a useful and important method of gene transfer, retroviral vectors can be genetically unstable. In the course of experiments using DOEJS, a retroviral vector able to confer expression of a H-ras oncogene and a neomycin resistance gene (neo) on mammalian cells (Compere et al., 1989), it was found that the vast majority of infected rat embryo fibroblasts, recovered on the basis of neo activity (i.e., G418 resistance), did not express ras mRNA. It was subsequently observed that most cells in the ψ2 cell line used to propagate DOEJS failed to produce virus capable of expressing both ras and neo in primary rat embryo fibroblasts. A simplified RNA extraction and slot-blot technique was developed to screen mRNA from several hundred fibroblast clones and, in doing so, infected fibroblast clones producing both neo and ras mRNA were identified at low frequency. The DOEJS/ψ2 packaging line was subsequently subcloned and individual clones screened for their ability to confer appropriate gene expression on target cells. Subclone DOEJS/ψ2-B6 was eventually isolated after screening 24 DOEJS subclones and 240 infected rat embryo fibroblast colonies. DOEJS/ψ2-B6 was shown to induce reliably phenotypic transformation, G418 resistance, and ras and neo mRNA expression in primary rat embryo fibroblasts. The RNA extraction and screening procedure was thus useful for recovering an infrequent subclone producing a retrovirus with the original properties.