Efficient screening of retroviral cDNA expression libraries.

Abstract

Expression cloning of cDNAs was first described a decade ago and was based on transient expression of cDNA libraries in COS cells. In contrast to transient transfection of plasmids, retroviral gene transfer delivers genes stably into a wide range of target cells. We utilize a simple packaging system for production of high-titer retrovirus stock from cDNA libraries to establish a cDNA expression cloning system. In two model experiments, murine interleukin (IL)-3-dependent Ba/F3 cells were infected with libraries of retrovirally expressed cDNA derived from human T-cell mRNA or human IL-3-dependent TF-1 cell line mRNA.These infected Ba/F3 cells were selected for the expression of CD2 by flow cytometry or for the a subunit of the human IL-3 receptor (hIL-3Ra) by factor-dependent growth. CD2 (frequency, 1 in 104) and hIL-3Ra (frequency, 1 in 1.5 x 105) cDNAs were readily detected in small-scale experiments, indicating this retroviral expression cloning system is efficient enough to clone low-abundance cDNAs by their expression or function.Expression cloning is a powerful tool with which to isolate a cDNA of interest when a phenotypic function of a protein is known but its amino acid sequence is not known. For instance, many cytokine cDNAs were cloned by assaying the growthpromoting activity of culture supematant of COS cells transiently transfected with subdivided cDNA libraries (1). The principle of the first cDNA expression cloning system (2) was to amplify expression vectors carrying the simian virus 40 (SV40) replication origin (ori) in mammalian cells stably expressing tumor antigen (T antigen) [i.e., a transformed African green monkey kidney cell line, COS (3)]. The presence of the SV40 large T antigen in COS cells allows replication of SV40 ori-containing plasmids, thus amplifying expression of the cDNA on the plasmid (2, 3). A notable alteration to this strategy was introduced by Seed and Aruffo (4), in which transfected COS cells were selected for the expression of a known antigen by panning transfected cells on antibody-coated plates. Genes encoding many surface markers for which antibodies were available have been cloned in this way (4,5). Alternatively, the cell sorter was also utilized to collect COS cells that express a cDNA of interest (6). The cDNAs for cytokine receptors have been also isolated from cDNA libraries by screening for cytokine binding (7-9). In addition, a transcription factor GATA-1 cDNA was isolated by COS-cell expression cloning using gel mobility shift assay as a screening procedure (10). Thus, expression cloning of genes expressing a desired protein or activity is limited only by the ingenuity of the experimental design.Despite many applications, most conventional expression cloning systems still suffer from the need for transient amplification of plasmids in particular cell lines expressing the SV40 (or polyoma) large T antigen. First of all, the function of the target gene has to be suited to transient detection. Moreover, target cells are restricted to cells that allow SV...

v-Src suppresses SHPS-1 expression via the Ras-MAP kinase pathway to promote the oncogenic growth of cells

“…For the expression of SHPS-1 gene, pBabe-puro vector or a retrovirus vector, pMx-puro (Kitamura et al, 1995), was employed. In case of pMx-puro, recombinant retroviruses were recovered from Bosc23 cells transfected with SHPS-1 gene and infected to SR3Y1 as described (Kitamura et al, 1995).…”

“…In case of pMx-puro, recombinant retroviruses were recovered from Bosc23 cells transfected with SHPS-1 gene and infected to SR3Y1 as described (Kitamura et al, 1995). A recombinant virus made by vector only was used as a control.…”

T Cell Expression Cloning of aMycobacterium tuberculosisGene Encoding a Protective Antigen Associated with the Early Control of Infection

“…Target cells were EL-4 cells retrovirally transduced with the MTB41 gene essentially as described (40). Briefly, the retroviral construct was used in transfections of Phoenix-Ampho, an amphotropic retroviral packaging line.…”

Production of complex nucleic acid libraries using highly parallel in situ oligonucleotide synthesis

“…Nucleic acid libraries provide some of the most versatile tools for functional analysis of genomes, individual proteins or complexes [1][2][3][4][5][6][7][8][9][10][11][12][13][14] . These libraries can be constructed using either biologically derived or chemically synthesized nucleic acids as substrates.…”