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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.


Working model for the recombination in FRET biosensors.(A) The recombination of FRET biosensors with h25-e75YPet generates CFP, which includes the critical amino acid substitution of Y66W from GFP. (B) The recombination of FRET biosensors with e75-h25YPet generates GFP or YFP, which includes the critical amino acid substitution of T203Y from GFP.
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f2: Working model for the recombination in FRET biosensors.(A) The recombination of FRET biosensors with h25-e75YPet generates CFP, which includes the critical amino acid substitution of Y66W from GFP. (B) The recombination of FRET biosensors with e75-h25YPet generates GFP or YFP, which includes the critical amino acid substitution of T203Y from GFP.

Mentions: Recombination of retroviral genomes could be caused by template switching/jumping during minus-strand DNA synthesis, or reverse transcription, on the RNA genome and plus-strand DNA replication. Figure 2 shows the case of template switching/jumping during reverse transcription. This model provided a proper interpretation of the results with the series of lentiviral or retroviral vectors: Assuming that e100YPet is not recombined with nTurquoise-GL and that the hues of YFP and CFP are determined primarily by the T203Y substitution and the Y66W substitution, respectively29, we can expect the outcomes of recombination between the YPet chimeras and nTurquoise-GL illustrated in Fig. 2: First, h25-e75YPet is recombined with nTurquoise-GL in the first-quarter segment. In this case, the recombined fluorescent protein gene will carry the nucleotides for Y66W, but not for T203Y, and therefore encodes CFP (Fig. 2A). Second, e75-h25YPet is recombined with nTurquoise-GL in the fourth-quarter segment. In this case, the resulting fluorescent protein will miss the nucleotides for Y66W and may or may not contain the nucleotides for T203Y, and therefore encodes either GFP or YFP (Fig. 2B). The recombination between YPet and nTurquoise-GL generate 4 types of chimeric GFP (Supplementary Fig. S2A and S2B). It is expected that chimeric GFP with H148G mutation fluoresced less than the wild-type GFP and YFP, because of the higher pKa value30. In fact, we confirmed that the chimeric GFP emitted less fluorescence than original YPet in our experimental condition (Supplementary Fig. S2B). Therefore, the decrease of fluorescence was caused by substation the nTurquoise-GL sequence for YPet sequence.


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)

Working model for the recombination in FRET biosensors.(A) The recombination of FRET biosensors with h25-e75YPet generates CFP, which includes the critical amino acid substitution of Y66W from GFP. (B) The recombination of FRET biosensors with e75-h25YPet generates GFP or YFP, which includes the critical amino acid substitution of T203Y from GFP.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Working model for the recombination in FRET biosensors.(A) The recombination of FRET biosensors with h25-e75YPet generates CFP, which includes the critical amino acid substitution of Y66W from GFP. (B) The recombination of FRET biosensors with e75-h25YPet generates GFP or YFP, which includes the critical amino acid substitution of T203Y from GFP.
Mentions: Recombination of retroviral genomes could be caused by template switching/jumping during minus-strand DNA synthesis, or reverse transcription, on the RNA genome and plus-strand DNA replication. Figure 2 shows the case of template switching/jumping during reverse transcription. This model provided a proper interpretation of the results with the series of lentiviral or retroviral vectors: Assuming that e100YPet is not recombined with nTurquoise-GL and that the hues of YFP and CFP are determined primarily by the T203Y substitution and the Y66W substitution, respectively29, we can expect the outcomes of recombination between the YPet chimeras and nTurquoise-GL illustrated in Fig. 2: First, h25-e75YPet is recombined with nTurquoise-GL in the first-quarter segment. In this case, the recombined fluorescent protein gene will carry the nucleotides for Y66W, but not for T203Y, and therefore encodes CFP (Fig. 2A). Second, e75-h25YPet is recombined with nTurquoise-GL in the fourth-quarter segment. In this case, the resulting fluorescent protein will miss the nucleotides for Y66W and may or may not contain the nucleotides for T203Y, and therefore encodes either GFP or YFP (Fig. 2B). The recombination between YPet and nTurquoise-GL generate 4 types of chimeric GFP (Supplementary Fig. S2A and S2B). It is expected that chimeric GFP with H148G mutation fluoresced less than the wild-type GFP and YFP, because of the higher pKa value30. In fact, we confirmed that the chimeric GFP emitted less fluorescence than original YPet in our experimental condition (Supplementary Fig. S2B). Therefore, the decrease of fluorescence was caused by substation the nTurquoise-GL sequence for YPet sequence.

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.