Improved biolistic transfection of hair cells.
Transient transfection of hair cells has proven challenging. Here we describe modifications to the Bio-Rad Helios Gene Gun that, along with an optimized protocol, improve transfection of bullfrog, chick, and mouse hair cells. The increased penetrating power afforded by our method allowed us to trans...
| Main Authors: | , , |
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| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2012-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC3462194?pdf=render |
| _version_ | 1818848218668072960 |
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| author | Hongyu Zhao Matthew R Avenarius Peter G Gillespie |
| author_facet | Hongyu Zhao Matthew R Avenarius Peter G Gillespie |
| author_sort | Hongyu Zhao |
| collection | DOAJ |
| description | Transient transfection of hair cells has proven challenging. Here we describe modifications to the Bio-Rad Helios Gene Gun that, along with an optimized protocol, improve transfection of bullfrog, chick, and mouse hair cells. The increased penetrating power afforded by our method allowed us to transfect mouse hair cells from the basal side, through the basilar membrane; this configuration protects hair bundles from damage during the procedure. We characterized the efficiency of transfection of mouse hair cells with fluorescently-tagged actin fusion protein using both the optimized procedure and a published procedure; while the efficiency of the two methods was similar, the morphology of transfected hair cells was improved with the new procedure. In addition, using the improved method, we were able to transfect hair cells in the bullfrog sacculus and chick cochlea for the first time. We used fluorescent-protein fusions of harmonin b (USH1C) and PMCA2 (ATP2B2; plasma-membrane Ca(2+)-ATPase isoform 2) to examine protein distribution in hair cells. While PMCA2-EGFP localization was similar to endogenous PMCA2 detected with antibodies, high levels of harmonin-EGFP were found at stereocilia tapers in bullfrog and chick, but not mouse; by contrast, harmonin-EGFP was concentrated in stereocilia tips in mouse hair cells. |
| first_indexed | 2024-12-19T06:13:51Z |
| format | Article |
| id | doaj.art-c0648dc13a3148fa94e8052613fec8f0 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-19T06:13:51Z |
| publishDate | 2012-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-c0648dc13a3148fa94e8052613fec8f02022-12-21T20:32:56ZengPublic Library of Science (PLoS)PLoS ONE1932-62032012-01-01710e4676510.1371/journal.pone.0046765Improved biolistic transfection of hair cells.Hongyu ZhaoMatthew R AvenariusPeter G GillespieTransient transfection of hair cells has proven challenging. Here we describe modifications to the Bio-Rad Helios Gene Gun that, along with an optimized protocol, improve transfection of bullfrog, chick, and mouse hair cells. The increased penetrating power afforded by our method allowed us to transfect mouse hair cells from the basal side, through the basilar membrane; this configuration protects hair bundles from damage during the procedure. We characterized the efficiency of transfection of mouse hair cells with fluorescently-tagged actin fusion protein using both the optimized procedure and a published procedure; while the efficiency of the two methods was similar, the morphology of transfected hair cells was improved with the new procedure. In addition, using the improved method, we were able to transfect hair cells in the bullfrog sacculus and chick cochlea for the first time. We used fluorescent-protein fusions of harmonin b (USH1C) and PMCA2 (ATP2B2; plasma-membrane Ca(2+)-ATPase isoform 2) to examine protein distribution in hair cells. While PMCA2-EGFP localization was similar to endogenous PMCA2 detected with antibodies, high levels of harmonin-EGFP were found at stereocilia tapers in bullfrog and chick, but not mouse; by contrast, harmonin-EGFP was concentrated in stereocilia tips in mouse hair cells.http://europepmc.org/articles/PMC3462194?pdf=render |
| spellingShingle | Hongyu Zhao Matthew R Avenarius Peter G Gillespie Improved biolistic transfection of hair cells. PLoS ONE |
| title | Improved biolistic transfection of hair cells. |
| title_full | Improved biolistic transfection of hair cells. |
| title_fullStr | Improved biolistic transfection of hair cells. |
| title_full_unstemmed | Improved biolistic transfection of hair cells. |
| title_short | Improved biolistic transfection of hair cells. |
| title_sort | improved biolistic transfection of hair cells |
| url | http://europepmc.org/articles/PMC3462194?pdf=render |
| work_keys_str_mv | AT hongyuzhao improvedbiolistictransfectionofhaircells AT matthewravenarius improvedbiolistictransfectionofhaircells AT peterggillespie improvedbiolistictransfectionofhaircells |