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Quantitative analysis of recombination between YFP and CFP genes of FRET biosensors introduced by lentiviral or retroviral gene transfer.

Komatsubara AT, Matsuda M, Aoki K - Sci Rep (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Bioimaging and Cell Signaling, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.

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.

No MeSH data available.


Computer simulation of recombination between the YFP and CFP genes.The recombination of YFP and CFP genes in A549 cells infected with the indicated lentivirus was simulated by computer with a recombination rate of 0.0043 (/base), which showed maximal likelihood estimation. To reproduce the experimental data, 9 parameters were extracted from the experimental data set in Fig. 3 (see Supplementary Fig. S6 and the Methods for details). Red lines were fitted with the e100YPet data. Orange and cyan arrowheads indicate the T203Y and Y66W positions, respectively.
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f6: Computer simulation of recombination between the YFP and CFP genes.The recombination of YFP and CFP genes in A549 cells infected with the indicated lentivirus was simulated by computer with a recombination rate of 0.0043 (/base), which showed maximal likelihood estimation. To reproduce the experimental data, 9 parameters were extracted from the experimental data set in Fig. 3 (see Supplementary Fig. S6 and the Methods for details). Red lines were fitted with the e100YPet data. Orange and cyan arrowheads indicate the T203Y and Y66W positions, respectively.

Mentions: The simple mathematical model qualitatively reproduced the four experimental data sets of A549 cells and HeLa cells infected with lentiviral or retroviral vectors (Fig. 6 and Supplementary Fig. S7). These data validated our hypothesis that the recombination was governed by a random process within the homology region. Interestingly, the recombination rate of lentivirus did not differ from that of retrovirus; both were 0.002 ~ 0.005 /base (Table 1 and Supplementary Fig. S8). Further, the lentivirus with shorter spacer between YFP and CFP genes indeed showed smaller recombination rate than that with longer spacer (Table 1).


Quantitative analysis of recombination between YFP and CFP genes of FRET biosensors introduced by lentiviral or retroviral gene transfer.

Komatsubara AT, Matsuda M, Aoki K - Sci Rep (2015)

Computer simulation of recombination between the YFP and CFP genes.The recombination of YFP and CFP genes in A549 cells infected with the indicated lentivirus was simulated by computer with a recombination rate of 0.0043 (/base), which showed maximal likelihood estimation. To reproduce the experimental data, 9 parameters were extracted from the experimental data set in Fig. 3 (see Supplementary Fig. S6 and the Methods for details). Red lines were fitted with the e100YPet data. Orange and cyan arrowheads indicate the T203Y and Y66W positions, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4542544&req=5

f6: Computer simulation of recombination between the YFP and CFP genes.The recombination of YFP and CFP genes in A549 cells infected with the indicated lentivirus was simulated by computer with a recombination rate of 0.0043 (/base), which showed maximal likelihood estimation. To reproduce the experimental data, 9 parameters were extracted from the experimental data set in Fig. 3 (see Supplementary Fig. S6 and the Methods for details). Red lines were fitted with the e100YPet data. Orange and cyan arrowheads indicate the T203Y and Y66W positions, respectively.
Mentions: The simple mathematical model qualitatively reproduced the four experimental data sets of A549 cells and HeLa cells infected with lentiviral or retroviral vectors (Fig. 6 and Supplementary Fig. S7). These data validated our hypothesis that the recombination was governed by a random process within the homology region. Interestingly, the recombination rate of lentivirus did not differ from that of retrovirus; both were 0.002 ~ 0.005 /base (Table 1 and Supplementary Fig. S8). Further, the lentivirus with shorter spacer between YFP and CFP genes indeed showed smaller recombination rate than that with longer spacer (Table 1).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Bioimaging and Cell Signaling, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.

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.

No MeSH data available.