Antiviral Hammerhead Ribozymes Are Effective for Developing Transgenic Suppression of Chikungunya Virus in Aedes aegypti Mosquitoes
Abstract
:1. Background
2. Materials and Method
2.1. Selection of hRz Target Sites in the CHIKV Genome
2.2. Plasmid Construction
2.3. Cell Cultures, Transfection, Clonal Generation and Infection
2.4. RT-PCR Analysis
2.5. TCID50 Immunofluorescence Assays
2.6. Caspase 3 Assays
2.7. Quantitative RT-PCR
2.8. Generation of hRz Transgenic Mosquito Lines
2.9. Splinkerette PCR
2.10. Mosquitoes, Infection and Maintenance
2.11. Genotyping of Mosquitoes
2.12. In Situ Indirect Immuno-Fluorescent Assays
2.13. Assays of Virus in Saliva
3. Results
3.1. Construction of Transformation Vectors Carrying Anti-CHIKV hRz
3.2. Single-Cell Sorting for Transformed Selected Cell Populations and Screening for CHIKV Suppression
3.3. TCID50-IFA Analysis of CHIKV Suppression in Clones Lacking Visible CPE
3.4. qRT-PCR Analysis of Ribozyme-Expressing Cell Clones Demonstrates Complete Suppression
3.5. Caspase 3 Assays of Ribozyme-Expressing Cell Clones Confirms Lack of CHIKV Infection
3.6. Construction of PiggyBac Vectors and Establishment of Transgenic Mosquitoes
3.7. RT-PCR to Confirm Transgene Expression
3.8. Identification of Each Transgene Integration Site via Splinkerette PCR
3.9. Analysis of CHIKV Infection Suppression in hRz Transgenic Mosquitoes
3.10. Direct PCR Analysis Confirms Haploid Genotype of G6 mosquitoes
3.11. CHIKV Titers in Saliva Collected from Challenged Transgenic Female Mosquitoes
4. Discussion
Supplementary Materials
Supplementary File 1Acknowledgments
Author Contributions
References
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hRz | Template Sequence | Target Sequence (5’–3’) |
---|---|---|
hRz#9 | cgaacccgggcactacaaaaaccaacaaCTATTTAGCTGATGAGGCCGAAAGGCCGAAACCGCCGTACgggcccgggcccaaaaa | gtacggcggtcctaaatag |
hRz#10 | cgaacccgggcactacaaaaaccaacaaAAGCGTCGCTGATGAGGCCGAAAGGCCGAAACTTCATGTGggggcccgggcccaaaaa | cacatgaagtccgacgctt |
hRz#11 | cgaacccgggcactacaaaaaccaacaaTTACGCGGCTGATGAGGCCGAAAGGCCGAAACCAGAGGGgggcccgggcccaaaaa | ccctctggtcccgcgtaa |
hRz#12 | cgaacccgggcactacaaaaaccaacaaAGCATGATCTGATGAGGCCGAAAGGCCGAAACTTGGTTTTggggcccgggcccaaaaa | aaaaccaagtcatcatgct |
hRz#13 | cgaacccgggcactacaaaaaccaacaaAATGGGTACTGATGAGGCCGAAAGGCCGAAACGCCGGTGAgggcccgggcccaaaaa | tcaccggcgtctacccatt |
hRz#14 | cgaacccgggcactacaaaaaccaacaaAGGCTGAACTGATGAGGCCGAAAGGCCGAAACATTGGCCCgggcccgggcccaaaaa | gggccaatgtcttcagcct |
hRz#15 | cgaacccgggcactacaaaaaccaacaaTCTTAGGGCTGATGAGGCCGAAAGGCCGAAACACATATACgggcccgggcccaaaaa | gtatatgtgtcccctaaga |
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Mishra, P.; Furey, C.; Balaraman, V.; Fraser, M.J. Antiviral Hammerhead Ribozymes Are Effective for Developing Transgenic Suppression of Chikungunya Virus in Aedes aegypti Mosquitoes. Viruses 2016, 8, 163. https://doi.org/10.3390/v8060163
Mishra P, Furey C, Balaraman V, Fraser MJ. Antiviral Hammerhead Ribozymes Are Effective for Developing Transgenic Suppression of Chikungunya Virus in Aedes aegypti Mosquitoes. Viruses. 2016; 8(6):163. https://doi.org/10.3390/v8060163
Chicago/Turabian StyleMishra, Priya, Colleen Furey, Velmurugan Balaraman, and Malcolm J. Fraser. 2016. "Antiviral Hammerhead Ribozymes Are Effective for Developing Transgenic Suppression of Chikungunya Virus in Aedes aegypti Mosquitoes" Viruses 8, no. 6: 163. https://doi.org/10.3390/v8060163