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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

Recombination between the YFP and CFP genes by lentiviral gene transfer.(A–H) A549 cells were infected with lentivirus encoding 8 different FRET biosensors as shown in Fig. 1B. At least 4 days after infection, the cells were imaged with an epi-fluorescence microscope. The average fluorescence intensities of CFP and YFP are represented as a log-log plot. Each dot corresponds to an A549 cell. Three hundred cells were analyzed from two independent experiments. Red lines are the fitted line with the e100YPet data. Orange and cyan arrowheads indicate the T203Y and Y66W positions, respectively.
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f3: Recombination between the YFP and CFP genes by lentiviral gene transfer.(A–H) A549 cells were infected with lentivirus encoding 8 different FRET biosensors as shown in Fig. 1B. At least 4 days after infection, the cells were imaged with an epi-fluorescence microscope. The average fluorescence intensities of CFP and YFP are represented as a log-log plot. Each dot corresponds to an A549 cell. Three hundred cells were analyzed from two independent experiments. Red lines are the fitted line with the e100YPet data. Orange and cyan arrowheads indicate the T203Y and Y66W positions, respectively.

Mentions: Most cells infected with the h100YPet-carrying lentivirus demonstrated clear evidence of recombination between the YFP and CFP genes, i.e., cells expressed either YFP or CFP, but not both (Fig. 3A). In cells infected with the h75-e25YPet-carrying virus, the fraction of cells emitting YFP fluorescence alone was smaller than that in h100YPet-carrying virus-infected cells, and cells emitting both CFP and YFP fluorescences were detectable (Fig. 3B). In cells infected with the h50-e50YPet-carrying virus or h25-e75YPet-carrying virus, all cells emitted either CFP fluorescence alone or both CFP and YFP fluorescences (Fig. 3C,D). As expected, the e100YPet-carrying lentivirus did not show any sign of recombination and emitted equal amounts of CFP and YFP fluorescence (Fig. 3E). In cells infected with the e75-h25YPet-carrying virus, a small fraction of cells emitted YFP fluorescence alone (Fig. 3F). The fraction of cells that emitted YFP fluorescence alone was increased in the e50-h50YPet-carrying virus and e25-h75YPet-carrying virus, concomitant with the decrease in cells emitting both CFP and YFP fluorescences (Fig. 3G,H). Of note, the fluorescence intensity of chimeric fluorescent proteins was less than the prototype as described; the average intensities of YFP channel was highest in the prototype e100YPet-carrying virus (Fig. 3E–H). Overall, very similar results were obtained when HeLa cells were infected at an MOI of less than 1.0 (Supplementary Fig. S3A–S3H). These results are in line with the model shown in Fig. 2, supporting the idea that the recombination is caused by template switching/jumping during reverse transcription.


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)

Recombination between the YFP and CFP genes by lentiviral gene transfer.(A–H) A549 cells were infected with lentivirus encoding 8 different FRET biosensors as shown in Fig. 1B. At least 4 days after infection, the cells were imaged with an epi-fluorescence microscope. The average fluorescence intensities of CFP and YFP are represented as a log-log plot. Each dot corresponds to an A549 cell. Three hundred cells were analyzed from two independent experiments. Red lines are the fitted line 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

f3: Recombination between the YFP and CFP genes by lentiviral gene transfer.(A–H) A549 cells were infected with lentivirus encoding 8 different FRET biosensors as shown in Fig. 1B. At least 4 days after infection, the cells were imaged with an epi-fluorescence microscope. The average fluorescence intensities of CFP and YFP are represented as a log-log plot. Each dot corresponds to an A549 cell. Three hundred cells were analyzed from two independent experiments. Red lines are the fitted line with the e100YPet data. Orange and cyan arrowheads indicate the T203Y and Y66W positions, respectively.
Mentions: Most cells infected with the h100YPet-carrying lentivirus demonstrated clear evidence of recombination between the YFP and CFP genes, i.e., cells expressed either YFP or CFP, but not both (Fig. 3A). In cells infected with the h75-e25YPet-carrying virus, the fraction of cells emitting YFP fluorescence alone was smaller than that in h100YPet-carrying virus-infected cells, and cells emitting both CFP and YFP fluorescences were detectable (Fig. 3B). In cells infected with the h50-e50YPet-carrying virus or h25-e75YPet-carrying virus, all cells emitted either CFP fluorescence alone or both CFP and YFP fluorescences (Fig. 3C,D). As expected, the e100YPet-carrying lentivirus did not show any sign of recombination and emitted equal amounts of CFP and YFP fluorescence (Fig. 3E). In cells infected with the e75-h25YPet-carrying virus, a small fraction of cells emitted YFP fluorescence alone (Fig. 3F). The fraction of cells that emitted YFP fluorescence alone was increased in the e50-h50YPet-carrying virus and e25-h75YPet-carrying virus, concomitant with the decrease in cells emitting both CFP and YFP fluorescences (Fig. 3G,H). Of note, the fluorescence intensity of chimeric fluorescent proteins was less than the prototype as described; the average intensities of YFP channel was highest in the prototype e100YPet-carrying virus (Fig. 3E–H). Overall, very similar results were obtained when HeLa cells were infected at an MOI of less than 1.0 (Supplementary Fig. S3A–S3H). These results are in line with the model shown in Fig. 2, supporting the idea that the recombination is caused by template switching/jumping during reverse transcription.

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