CONSTRUCTION AND CHARACTERIZATION OF A SELECTABLE MULTIDRUG-RESISTANCE GLUCOCEREBROSIDASE FUSION GENE

Gene fusions can be employed to ensure concomitant expression of two d ifferent proteins under the same transcriptional control elements, We have synthesized a retroviral expression vector (pHaMG1) containing a human multidrug resistance (MDR1)-glucocerebrosidase (GC) chimeric gen e inserted between the long terminal repeats of the Harvey murine sarc oma virus, When introduced into psi-CRE mouse fibroblasts, pHaMG1 conf erred the drug-selectable multidrug resistance phenotype, and drug-res istant clones produced active human GC of about 60 kDa, Percoll gradie nt fractionation of homogenates prepared from transfectants confirmed correct targeting of P-glycoprotein to the plasma membrane and of GC t o lysosomes, Although this construction was designed as a translationa l fusion of the MDR1 gene product, P-glycoprotein, and human GC, no ev idence for a fusion protein was found in transfected cells, and an ana lysis of the RNAs transcribed from the integrated pHaMG1 retroviral ve ctor suggests that either P-glycoprotein and GC are translated from on e mRNA and rapidly processed into two proteins or they are translated separately from different mRNAs, These results reveal the feasibility of using fusion genes, which are smaller than alternative construction s with two promoters or with an internal ribosome entry site, for coex pression of selectable and nonselectable cDNAs In retroviral vectors.