APPLICATIONS OF RETROVIRUS-MEDIATED EXPRESSION CLONING

We have recently established a novel expression cloning system using r etroviral vectors. The system is based on a high-efficiency packaging cell line, BOSC23, and a simplified retroviral vector, pBabeX, carryin g no selection marker. cDNA libraries, constructed in the pBabeX vecto r, are transiently transfected into BOSC23 cells. The supernatant cont ains more than 3x10(6)/mL, which would cover large complexities of cDN A libraries. The retrovirus stock gave 100% infection efficiency in NI H3T3 cells and 5-40% infection efficiency in various hematopoietic cel l lines. in contrast to the conventional expression cloning system, in which it is necessary to transfect cDNA libraries transiently into pa rticular cell types such as COS cells, retrovirus-mediated expression cloning allows us to transduce cDNAs into a wide variety of cell types . This method therefore makes it possible to select cells expressing a cDNA of interest by various functional assays. When combined with pol ymerase chain reaction (PCR)-driven random mutagenesis, this system is also useful in searching for mutations of various molecules that will result in alterations of their functions.