A NEW RETROVIRAL VECTOR, CA1, TO IDENTIFY AND SELECT FOR CELLS EXPRESSING AN INSERTED GENE IN-VITRO AND IN-VIVO

A new retroviral vector has been constructed that expresses genes enco ding three different activities from a single transcript. This feature has been exploited to enable the efficient marking and selection of c ells that express a gene of interest. The marker gene lacZ, encoding b eta-galactosidase, and neo, encoding neomycin phosphotransferase, for selection by the antibiotic G418, are expressed as a fusion, beta Geo. The expression of beta Geo is coordinated with expression of a gene o f interest at the mRNA level using an Internal Ribosome Entry Site (IR ES) from the Encephalomyocarditis Virus (EMCV). The IRES promotes cap- independent initiation of translation therefore two reading frames can be translated from a single transcript. In vitro, the vector has been shown to confer beta-galactosidase activity, transformation by v-src and resistance to G418, following infection of cells. To show that the retrovirus was able to mark infected cells in vivo, cells infected wi th the retrovirus were transplanted into mouse mammary gland where the y grew and were successfully located by staining for beta-galactosidas e over 2 months after transplantation. (C) 1997 Elsevier Science B.V.