Harnessing Intronic microRNA Structures to Improve Tolerance and Expression of shRNAs in Animal Cells
Exogenous RNA polymerase III (pol III) promoters are commonly used to express short hairpin RNA (shRNA). Previous studies have indicated that expression of shRNAs using standard pol III promoters can cause toxicity in vivo due to saturation of the native miRNA pathway. A potential way of mitigating...
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MDPI AG
2022-02-01
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| Series: | Methods and Protocols |
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| Online Access: | https://www.mdpi.com/2409-9279/5/1/18 |
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| author | Arjun Challagulla Mark L. Tizard Timothy J. Doran David M. Cahill Kristie A. Jenkins |
| author_facet | Arjun Challagulla Mark L. Tizard Timothy J. Doran David M. Cahill Kristie A. Jenkins |
| author_sort | Arjun Challagulla |
| collection | DOAJ |
| description | Exogenous RNA polymerase III (pol III) promoters are commonly used to express short hairpin RNA (shRNA). Previous studies have indicated that expression of shRNAs using standard pol III promoters can cause toxicity in vivo due to saturation of the native miRNA pathway. A potential way of mitigating shRNA-associated toxicity is by utilising native miRNA processing enzymes to attain tolerable shRNA expression levels. Here, we examined parallel processing of exogenous shRNAs by harnessing the natural miRNA processing enzymes and positioning a shRNA adjacent to microRNA107 (miR107), located in the intron 5 of the Pantothenate Kinase 1 (PANK1) gene. We developed a vector encoding the PANK1 intron containing miR107 and examined the expression of a single shRNA or multiple shRNAs. Using qRT-PCR analysis and luciferase assay-based knockdown assay, we confirmed that miR30-structured shRNAs have resulted in the highest expression and subsequent transcript knockdown. Next, we injected Hamburger and Hamilton stage 14–15 chicken embryos with a vector encoding multiple shRNAs and confirmed that the parallel processing was not toxic. Taken together, this data provides a novel strategy to harness the native miRNA processing pathways for shRNA expression. This enables new opportunities for RNAi based applications in animal species such as chickens. |
| first_indexed | 2024-03-09T21:20:04Z |
| format | Article |
| id | doaj.art-a9442f14327143e8b6319f4e51126b88 |
| institution | Directory Open Access Journal |
| issn | 2409-9279 |
| language | English |
| last_indexed | 2024-03-09T21:20:04Z |
| publishDate | 2022-02-01 |
| publisher | MDPI AG |
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| series | Methods and Protocols |
| spelling | doaj.art-a9442f14327143e8b6319f4e51126b882023-11-23T21:24:33ZengMDPI AGMethods and Protocols2409-92792022-02-01511810.3390/mps5010018Harnessing Intronic microRNA Structures to Improve Tolerance and Expression of shRNAs in Animal CellsArjun Challagulla0Mark L. Tizard1Timothy J. Doran2David M. Cahill3Kristie A. Jenkins4CSIRO Health and Biosecurity, Australian Centre for Disease Preparedness, Geelong, VIC 3219, AustraliaCSIRO Health and Biosecurity, Australian Centre for Disease Preparedness, Geelong, VIC 3219, AustraliaCSIRO Health and Biosecurity, Australian Centre for Disease Preparedness, Geelong, VIC 3219, AustraliaSchool of Life and Environmental Sciences, Deakin University, Geelong, VIC 3217, AustraliaCSIRO Health and Biosecurity, Australian Centre for Disease Preparedness, Geelong, VIC 3219, AustraliaExogenous RNA polymerase III (pol III) promoters are commonly used to express short hairpin RNA (shRNA). Previous studies have indicated that expression of shRNAs using standard pol III promoters can cause toxicity in vivo due to saturation of the native miRNA pathway. A potential way of mitigating shRNA-associated toxicity is by utilising native miRNA processing enzymes to attain tolerable shRNA expression levels. Here, we examined parallel processing of exogenous shRNAs by harnessing the natural miRNA processing enzymes and positioning a shRNA adjacent to microRNA107 (miR107), located in the intron 5 of the Pantothenate Kinase 1 (PANK1) gene. We developed a vector encoding the PANK1 intron containing miR107 and examined the expression of a single shRNA or multiple shRNAs. Using qRT-PCR analysis and luciferase assay-based knockdown assay, we confirmed that miR30-structured shRNAs have resulted in the highest expression and subsequent transcript knockdown. Next, we injected Hamburger and Hamilton stage 14–15 chicken embryos with a vector encoding multiple shRNAs and confirmed that the parallel processing was not toxic. Taken together, this data provides a novel strategy to harness the native miRNA processing pathways for shRNA expression. This enables new opportunities for RNAi based applications in animal species such as chickens.https://www.mdpi.com/2409-9279/5/1/18avianRNAipromoterstransgenicmiRNAsshRNA |
| spellingShingle | Arjun Challagulla Mark L. Tizard Timothy J. Doran David M. Cahill Kristie A. Jenkins Harnessing Intronic microRNA Structures to Improve Tolerance and Expression of shRNAs in Animal Cells Methods and Protocols avian RNAi promoters transgenic miRNAs shRNA |
| title | Harnessing Intronic microRNA Structures to Improve Tolerance and Expression of shRNAs in Animal Cells |
| title_full | Harnessing Intronic microRNA Structures to Improve Tolerance and Expression of shRNAs in Animal Cells |
| title_fullStr | Harnessing Intronic microRNA Structures to Improve Tolerance and Expression of shRNAs in Animal Cells |
| title_full_unstemmed | Harnessing Intronic microRNA Structures to Improve Tolerance and Expression of shRNAs in Animal Cells |
| title_short | Harnessing Intronic microRNA Structures to Improve Tolerance and Expression of shRNAs in Animal Cells |
| title_sort | harnessing intronic microrna structures to improve tolerance and expression of shrnas in animal cells |
| topic | avian RNAi promoters transgenic miRNAs shRNA |
| url | https://www.mdpi.com/2409-9279/5/1/18 |
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