Quantitative analysis of recombination between YFP and CFP genes of FRET biosensors introduced by lentiviral or retroviral gene transfer.
Biosensors based on the principle of Förster (or fluorescence) resonance energy transfer (FRET) have been developed to visualize spatio-temporal dynamics of signalling molecules in living cells. Many of them adopt a backbone of intramolecular FRET biosensor with a cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) as donor and acceptor, respectively. However, there remains the difficulty of establishing cells stably expressing FRET biosensors with a YFP and CFP pair by lentiviral or retroviral gene transfer, due to the high incidence of recombination between YFP and CFP genes. To address this, we examined the effects of codon-diversification of YFP on the recombination of FRET biosensors introduced by lentivirus or retrovirus. The YFP gene that was fully codon-optimized to E.coli evaded the recombination in lentiviral or retroviral gene transfer, but the partially codon-diversified YFP did not. Further, the length of spacer between YFP and CFP genes clearly affected recombination efficiency, suggesting that the intramolecular template switching occurred in the reverse-transcription process. The simple mathematical model reproduced the experimental data sufficiently, yielding a recombination rate of 0.002-0.005 per base. Together, these results show that the codon-diversified YFP is a useful tool for expressing FRET biosensors by lentiviral or retroviral gene transfer.
Laboratory of Bioimaging and Cell Signaling, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.
Akira T
AT
Komatsubara
Laboratory of Bioimaging and Cell Signaling, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.|Department of Pathology and Biology of Diseases, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.
Michiyuki
M
Matsuda
Imaging Platform for Spatio-Temporal Information, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.
Kazuhiro
K
Aoki
D001426:Bacterial Proteins / Q000378:metabolism*; D015374:Biosensing Techniques; D003062:Codon / Q000235:genetics; D003198:Computer Simulation; D031541:Fluorescence Resonance Energy Transfer; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D016086:Lentivirus / Q000378:metabolism*; D008164:Luminescent Proteins / Q000378:metabolism*; D008962:Models, Theoretical; D011995:Recombination, Genetic; D015203:Reproducibility of Results; D012190:Retroviridae / Q000378:metabolism*; D013698:Templates, Genetic
D001426:Bacterial Proteins; D003062:Codon; C485182:Cyan Fluorescent Protein; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins