Abstract
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.
Highlights
Biosensors based on the principle of Förster resonance energy transfer (FRET) have been developed to visualize spatio-temporal dynamics of signalling molecules in living cells
We demonstrated that a FRET biosensor comprised of e100YPet and nTurquoise-GL could be applied to lentivirus- or retrovirus-mediated gene transfer, and such transfer had the lowest possibility of recombination when performed in HeLa cells and A549 cells
Previous studies have proven the impact of codon usage on the gene expression[31], and we expected that the substitution of h100YPet partly or totally by e100YPet would decrease the expression of the FRET biosensor
Summary
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. 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. Most intramolecular FRET biosensors adopt a cyan fluorescent protein (CFP) and a yellow fluorescent protein (YFP) as the donor and acceptor fluorescent proteins, respectively Because both CFP and YFP are derived from GFP, they share high nucleotide sequence homology, which presumably causes the recombination. Further evidence is provided by the results that FRET biosensors carrying a YFP and a coral-derived teal fluorescent protein (TFP) are readily expressed by retrovirus-mediated gene transfer without any recombination[20,21,22]. TFP has some advantages over CFP as a FRET donor[23], the substitution of CFP to TFP decreases FRET gain more than in most FRET biosensors containing YFP as the acceptor, which could be due to the weak or absent dimerization of YFP and TFP13
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