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. 1997 Mar 18;94(6):2735–2740. doi: 10.1073/pnas.94.6.2735

Figure 1.

Figure 1

Construction of a recombinant retrovirus containing the coding region of the hTGFα gene under the control of the constitutively expressed mammalian phosphoglycerate kinase (PGK) promoter. An EcoRI–BalI cDNA fragment containing the entire coding region of hTGFα without the polyadenylylation signal (A) was subcloned into the EcoRI–HindIII sites of pBluescript (after blunting the HindIII site) (B). A BamHI–XhoI fragment was then subcloned into the multiple cloning site of PGKIRES (C) between the PGK promoter and the internal ribosomal entry site from the encephalomyocarditis virus (emcIRES), which allows efficient translation of both the hTGFα gene and the downstream neomycin-resistance gene. (D) Expression of hTGFα mRNA in transfected ψ CRIP cells and infected 3T3 cells (lane 1, DNA markers; lane 2, undigested probe; lane 3, nontransfected CRIP cells; lane 4, CRIP cells transfected with PGKIRES-hTGFα cDNA; lane 5, noninfected 3T3 cells; lane 6, 3T3 cells infected with PGKIRES-hTGFα viruses). (E) Release of TGFα from a colony of infected 3T3 cells into the culture medium.