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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 of FRET biosensors during lentiviral or retroviral infection.(A) Schematic representation of the recombination between YFP and CFP genes in FRET biosensors in the process of lentiviral or retroviral gene transfer. Two copackaged genomic RNAs encoding FRET biosensors are included in a virus particle. After infection, cells express only YFP or CFP. (B) FRET biosensors with different YFP variants. A PKA FRET biosensor, AKAR3EV, is composed of YPet (YFP), a FHA1 domain, linker, PKA substrate, nTurquoise-GL (CFP), and a nuclear export sequence (NES). In this study, YPet is replaced with h100YPet, H75-e25YPet, h50-e50YPet, h25-e75YPet, e100YPet, e75-h25YPet, e50-h50YPet, and e25-h75YPet.
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f1: Recombination of FRET biosensors during lentiviral or retroviral infection.(A) Schematic representation of the recombination between YFP and CFP genes in FRET biosensors in the process of lentiviral or retroviral gene transfer. Two copackaged genomic RNAs encoding FRET biosensors are included in a virus particle. After infection, cells express only YFP or CFP. (B) FRET biosensors with different YFP variants. A PKA FRET biosensor, AKAR3EV, is composed of YPet (YFP), a FHA1 domain, linker, PKA substrate, nTurquoise-GL (CFP), and a nuclear export sequence (NES). In this study, YPet is replaced with h100YPet, H75-e25YPet, h50-e50YPet, h25-e75YPet, e100YPet, e75-h25YPet, e50-h50YPet, and e25-h75YPet.

Mentions: We used a FRET biosensor for protein kinase A, AKAR3EV13, to examine the contribution of nucleotide sequence homology in recombination between CFP and YFP (Fig. 1A). AKAR3EV comprised a YFP-derived YPet27, and a CFP-derived nTurquoise-GL28 as the acceptor and donor, respectively. These fluorescent proteins sandwich the phosphate-binding domain of FHA1, EV linker, and a substrate peptide of PKA (Fig. 1B). The nuclear export signal (NES) was included at the C-terminus of the biosensor. Both the YPet and the nTurquoise-GL genes have been codon-optimized for humans. The homology between the humanized YPet, called hYPet hereafter, and nTurquoise-GL was 96%. As the codon-diversified YFP, we chose a YPet gene optimized for E. coli, called eYPet hereafter. The nucleotide sequence homology between eYPet and nTurquoise-GL was 68% (Supplementary Fig. S1). We constructed six YPet chimeras between hYPet and eYPet: h75-e25YPet, h50-e50YPet, h25-e75YPet, e75-h25YPet, e50-h50YPet, and e25-h75YPet (Fig. 1B, and see Methods). The order of h and e and their numbers indicated the order and the percentage ratio of hYPet to eYPet, respectively. For instance, h75-e25YPet was composed of the first 75% of the hYPet gene DNA sequence, followed by the last 25% of the eYPet gene DNA sequence. h100YPet and e100YPet are identical to the authentic hYPet and eYPet, respectively. These genes for FRET biosensors were inserted into either the MuLV-derived retroviral vector or HIV-derived lentiviral vector, which were transfected into 293T cells to generate retroviral or lentiviral vectors.


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 of FRET biosensors during lentiviral or retroviral infection.(A) Schematic representation of the recombination between YFP and CFP genes in FRET biosensors in the process of lentiviral or retroviral gene transfer. Two copackaged genomic RNAs encoding FRET biosensors are included in a virus particle. After infection, cells express only YFP or CFP. (B) FRET biosensors with different YFP variants. A PKA FRET biosensor, AKAR3EV, is composed of YPet (YFP), a FHA1 domain, linker, PKA substrate, nTurquoise-GL (CFP), and a nuclear export sequence (NES). In this study, YPet is replaced with h100YPet, H75-e25YPet, h50-e50YPet, h25-e75YPet, e100YPet, e75-h25YPet, e50-h50YPet, and e25-h75YPet.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Recombination of FRET biosensors during lentiviral or retroviral infection.(A) Schematic representation of the recombination between YFP and CFP genes in FRET biosensors in the process of lentiviral or retroviral gene transfer. Two copackaged genomic RNAs encoding FRET biosensors are included in a virus particle. After infection, cells express only YFP or CFP. (B) FRET biosensors with different YFP variants. A PKA FRET biosensor, AKAR3EV, is composed of YPet (YFP), a FHA1 domain, linker, PKA substrate, nTurquoise-GL (CFP), and a nuclear export sequence (NES). In this study, YPet is replaced with h100YPet, H75-e25YPet, h50-e50YPet, h25-e75YPet, e100YPet, e75-h25YPet, e50-h50YPet, and e25-h75YPet.
Mentions: We used a FRET biosensor for protein kinase A, AKAR3EV13, to examine the contribution of nucleotide sequence homology in recombination between CFP and YFP (Fig. 1A). AKAR3EV comprised a YFP-derived YPet27, and a CFP-derived nTurquoise-GL28 as the acceptor and donor, respectively. These fluorescent proteins sandwich the phosphate-binding domain of FHA1, EV linker, and a substrate peptide of PKA (Fig. 1B). The nuclear export signal (NES) was included at the C-terminus of the biosensor. Both the YPet and the nTurquoise-GL genes have been codon-optimized for humans. The homology between the humanized YPet, called hYPet hereafter, and nTurquoise-GL was 96%. As the codon-diversified YFP, we chose a YPet gene optimized for E. coli, called eYPet hereafter. The nucleotide sequence homology between eYPet and nTurquoise-GL was 68% (Supplementary Fig. S1). We constructed six YPet chimeras between hYPet and eYPet: h75-e25YPet, h50-e50YPet, h25-e75YPet, e75-h25YPet, e50-h50YPet, and e25-h75YPet (Fig. 1B, and see Methods). The order of h and e and their numbers indicated the order and the percentage ratio of hYPet to eYPet, respectively. For instance, h75-e25YPet was composed of the first 75% of the hYPet gene DNA sequence, followed by the last 25% of the eYPet gene DNA sequence. h100YPet and e100YPet are identical to the authentic hYPet and eYPet, respectively. These genes for FRET biosensors were inserted into either the MuLV-derived retroviral vector or HIV-derived lentiviral vector, which were transfected into 293T cells to generate retroviral or lentiviral vectors.

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