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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.


Related in: MedlinePlus

Effect of a short spacer between YFP and CFP on recombination.(A) Schematic representation of a FRET biosensor with YPet, GGSGG linker, nTurquoise-GL, and NES. (B,C) HeLa cells were infected with lentivirus encoding FRET biosensor with a short spacer, 15 bases (B) and full spacer, 812 bases (C). At least 4 days after infection, 300 cells were imaged with an epi-fluorescence microscope, and represented as in Fig. 3. Note that panel C is the same graph as in Supplementary Fig. 2A. Orange and cyan arrowheads indicate the T203Y and Y66W positions, respectively.
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f5: Effect of a short spacer between YFP and CFP on recombination.(A) Schematic representation of a FRET biosensor with YPet, GGSGG linker, nTurquoise-GL, and NES. (B,C) HeLa cells were infected with lentivirus encoding FRET biosensor with a short spacer, 15 bases (B) and full spacer, 812 bases (C). At least 4 days after infection, 300 cells were imaged with an epi-fluorescence microscope, and represented as in Fig. 3. Note that panel C is the same graph as in Supplementary Fig. 2A. Orange and cyan arrowheads indicate the T203Y and Y66W positions, respectively.

Mentions: We next examined whether the length of the spacer between YFP and CFP affected the recombination. For this purpose, we developed an h100YPet-carrying lentiviral vector, in which h100YPet and nTurquoise-GL were linked with a GGSGG linker (15 bases) (Fig. 5A). Cells infected with the h100YPet-GGSGG-nTurquoise-GL-carrying lentivirus exhibited markedly less, but not negligible, recombination (Fig. 5B) than did those infected with the h100YPet–carrying lentivirus, in which the length between the two fluorescent genes was 812 bases (Fig. 5C). This result may suggest that the recombination took place in a single copy of the lentiviral gene, rather than between the 2 copies of the lentiviral gene in a single virus (see Discussion).


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)

Effect of a short spacer between YFP and CFP on recombination.(A) Schematic representation of a FRET biosensor with YPet, GGSGG linker, nTurquoise-GL, and NES. (B,C) HeLa cells were infected with lentivirus encoding FRET biosensor with a short spacer, 15 bases (B) and full spacer, 812 bases (C). At least 4 days after infection, 300 cells were imaged with an epi-fluorescence microscope, and represented as in Fig. 3. Note that panel C is the same graph as in Supplementary Fig. 2A. 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

f5: Effect of a short spacer between YFP and CFP on recombination.(A) Schematic representation of a FRET biosensor with YPet, GGSGG linker, nTurquoise-GL, and NES. (B,C) HeLa cells were infected with lentivirus encoding FRET biosensor with a short spacer, 15 bases (B) and full spacer, 812 bases (C). At least 4 days after infection, 300 cells were imaged with an epi-fluorescence microscope, and represented as in Fig. 3. Note that panel C is the same graph as in Supplementary Fig. 2A. Orange and cyan arrowheads indicate the T203Y and Y66W positions, respectively.
Mentions: We next examined whether the length of the spacer between YFP and CFP affected the recombination. For this purpose, we developed an h100YPet-carrying lentiviral vector, in which h100YPet and nTurquoise-GL were linked with a GGSGG linker (15 bases) (Fig. 5A). Cells infected with the h100YPet-GGSGG-nTurquoise-GL-carrying lentivirus exhibited markedly less, but not negligible, recombination (Fig. 5B) than did those infected with the h100YPet–carrying lentivirus, in which the length between the two fluorescent genes was 812 bases (Fig. 5C). This result may suggest that the recombination took place in a single copy of the lentiviral gene, rather than between the 2 copies of the lentiviral gene in a single virus (see Discussion).

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.


Related in: MedlinePlus