BACKGROUND: In the past three decades, much advancement has been made in γ-retroviral vector mediated gene transfer. One widely used vector design is based on the MFG vector, which uses the Moloney murine leukemia virus (MoMLV) transcriptional unit with extended packaging signals and insertion of the native MoMLV envelope splice acceptor region immediate 5' to the gene of interest inserted at an NcoI restriction site, which contains a translation start codon. Little is known about the impact of variations in start codon location within MFG-based vectors on protein expression. METHODS: To evaluate variation in start condo placement, a gene encoding a T-cell receptor (TCR) was cloned into an MFG-based vector and site-directed mutagenesis was used to move the gene away from the splice acceptor, as well as alter the frame with respect to the upstream start codon. Kozak consensus sequences were also added to the gene in an attempt to improve translation. RESULTS: Protein expression as measured by TCR surface expression and biological activity was substantially reduced when the gene was placed downstream and out-of-frame with the NcoI start codon. Expression was reestablished by mutation of the upstream start site, although at a reduced level. These findings were repeated with two other genes, a dominant negative TGFβRII and the reporter protein dEGFP. CONCLUSIONS: These finding emphasize the scanning rule for translation initiation and stress the importance of cloning genes of interest into or near the native NcoI start site of MFG-based retroviral vectors.
BACKGROUND: In the past three decades, much advancement has been made in γ-retroviral vector mediated gene transfer. One widely used vector design is based on the MFG vector, which uses the Moloney murine leukemia virus (MoMLV) transcriptional unit with extended packaging signals and insertion of the native MoMLV envelope splice acceptor region immediate 5' to the gene of interest inserted at an NcoI restriction site, which contains a translation start codon. Little is known about the impact of variations in start codon location within MFG-based vectors on protein expression. METHODS: To evaluate variation in start condo placement, a gene encoding a T-cell receptor (TCR) was cloned into an MFG-based vector and site-directed mutagenesis was used to move the gene away from the splice acceptor, as well as alter the frame with respect to the upstream start codon. Kozak consensus sequences were also added to the gene in an attempt to improve translation. RESULTS: Protein expression as measured by TCR surface expression and biological activity was substantially reduced when the gene was placed downstream and out-of-frame with the NcoI start codon. Expression was reestablished by mutation of the upstream start site, although at a reduced level. These findings were repeated with two other genes, a dominant negative TGFβRII and the reporter protein dEGFP. CONCLUSIONS: These finding emphasize the scanning rule for translation initiation and stress the importance of cloning genes of interest into or near the native NcoI start site of MFG-based retroviral vectors.