High-efficiency identification of genes by functional analysis from a retroviral cDNA expression library

Abstract

Retroviral gene transfer efficiently delivers genes of interest stably into target cells, and expression cDNA cloning has been shown to be highly successful. Considering these two advantages, we now report a method by which one can identify genes stimulating cell growth through functional analysis. The first step requires the construction of a retroviral cDNA expression library and the optimization of transfection of vector DNA into virus packaging cells. The second step involves the cocultivation of target cells with libraries of retrovirusproducing cells, resulting in the amplification of target cells transduced with a gene(s) stimulating cell growth. Under standardized conditions of transfection, we detected an average of 4,000 independent clones per dish, among which expression of a retroviral 13-galactosidase gene at an abundance of 0.2% could be detected. Next, we demonstrated the augmentation of the sensitivity of the assay by retroviral infection and functional analysis. We did this by cocultivating factor-dependent (FD) cells with dishes of GP/E cells transfected with plasmids containing various molar ratios of pN2-IL3 DNA and retroviral library cDNA and by determining the highest dilution of pN2-1L3 which still resulted in the conversion of FD cells to factor independence. The retroviral interleukin-3 gene at an abundance as low as 0.001% could be detected. Indeed, we were able to detect from FD cells the development of factor-independent colonies with different phenotypes after retroviral transfer of cDNAs from an immortalized hemopoietic stem cell line. Thus, the combination of a standardized highefficiency DNA transfection and retrovirus-mediated gene transfer should facilitate the identification of genes capable of conferring to target FD cells a detectable new function or phenotype. By scaling up the size of the experiment realistically during screening, the assay can detect cDNA at an abundance of lower than 0.0001%.

The pCL vector system: rapid production of helper-free, high-titer, recombinant retroviruses

“…All supernatants were free of helper virus when measured by four different methods, including the most sensitive SC1 amplification-reverse transcriptase assay ( Table 2). The pCL vector system is well suited for rapid and efficient construction of retrovirus cDNA expression libraries, as it avoids genetic bottlenecks and ping-pong amplification steps that impose limitations on current systems that rely on stable retrovirus packaging cell lines (11,29). In addition, the high levels of packageable RNA produced by the pCL vectors make them well suited for shortening the time required to produce high-titer, pantropic retrovirus vectors (2), from 2 months to potentially less than a week.…”

Cloning of a G‐protein‐coupled receptor that shows an activity to transform NIH3T3 cells and is expressed in gastric cancer cells

“…Because the retroviral vector integrates into the host chromosome in a single copy, this enables screening for the desired cDNA clone without repeating the first screening steps. 8) This retroviral cDNA library was used to transduce NIH3T3 fibroblasts. Many colonies of cells that had lost contact inhibition were purified and expanded.…”

Restoration of lipopolysaccharide-mediated B-cell response after expression of a cDNA encoding a GTP-binding protein

“…Extraction of genomic DNA and total RNA was done according to the method described by Sambrook et al (22). The construction of the cDNA library was as described previously (35) and was modified from the method of Okayama and Berg (19). The source of mRNA for the cDNA library derived from LPS-stimulated spleen cells was obtained from C3H/HeOuJ mice.…”