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The position of the AUG start codon in MFG‐based γ‐retroviral vectors has a dramatic effect on translation‐dependent protein expression
Author(s) -
Frankel Timothy L.,
Zhang Ling,
Burns William R.,
Zheng Zhili,
Morgan Richard A.
Publication year - 2011
Publication title -
the journal of gene medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.689
H-Index - 91
eISSN - 1521-2254
pISSN - 1099-498X
DOI - 10.1002/jgm.1599
Subject(s) - start codon , biology , gene , stop codon , genetics , translation (biology) , eukaryotic translation , expression vector , vector (molecular biology) , viral vector , rna splicing , messenger rna , rna , recombinant dna
Abstract 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 Nco I 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 Nco I 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 Nco I start site of MFG‐based retroviral vectors. Copyright © 2011 John Wiley & Sons, Ltd.