The isolation and identification of cDNA genes by their heterologous expression and function.
Functional cDNA genes for cell surface proteins, receptors, growth factors and nuclear and cytoplasmic proteins can be isolated by a heterologous expression-function gene isolation strategy. The method of this strategy is dependent on highly efficient mammalian expression vectors, the properties of COS cells as hosts for SV40-origin-containing mammalian expression vectors, the well-established technology of constructing highly complex plasmid cDNA libraries and on the sensitivity of biological assays. These technologies are combined to identify a specific gene sequence by its biological phenotype. In theory this can be accomplished by individually testing each single gene of the cDNA library by expression in COS cells. In practice complex cDNA libraries are formed so that multiple cDNA genes can be tested and analyzed simultaneously. When the biological assay can discriminate phenotypic changes at the single cell level as typified by cellular morphology, cell membrane proteins or receptors or intracellular enzymes, then a reiterative expression screening method can be used. This was demonstrated by the isolation of cDNA genes encoding cell membrane proteins (7-10, 12). For the isolation of cDNA genes for growth factors and cytoplasmic enzymes by the expression-function cDNA gene isolation strategy, an alternative method is followed. The cDNA library is partitioned into sets of cDNA clones for transfection and analysis. The number of clones in a set is dependent on the efficiency of the expression vector used and on the sensitivity of the biological assay. For secreted cytokines and growth factors which can be simply detected by factor dependent cell lines, about 1,000 cDNA clones can be tested as a single set. By this strategy the genes for numerous hematopoietic growth factors, cytokines and interleukins have been isolated. With such numerous successes we can expect that the heterologous expression-function cDNA gene isolation strategy will become an important molecular biology method.
Wong
G G
GG
Genetics Institute, Inc., Cambridge, MA 02140-2387.
D000818:Animals; D002460:Cell Line; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D005786:Gene Expression Regulation; D015723:Gene Library; D005796:Genes; D014162:Transfection
D004247:DNA