Compact Bidirectional Promoters for Dual-Gene Expression in a Sleeping Beauty Transposon
Promoter choice is an essential consideration for transgene expression in gene therapy. The expression of multiple genes requires ribosomal entry or skip sites, or the use of multiple promoters. Promoter systems comprised of two separate, divergent promoters may significantly increase the size of ge...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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MDPI AG
2020-12-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/21/23/9256 |
| _version_ | 1797545773113540608 |
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| author | Kevin He S. M. Ali Hosseini Rad Aarati Poudel Alexander Donald McLellan |
| author_facet | Kevin He S. M. Ali Hosseini Rad Aarati Poudel Alexander Donald McLellan |
| author_sort | Kevin He |
| collection | DOAJ |
| description | Promoter choice is an essential consideration for transgene expression in gene therapy. The expression of multiple genes requires ribosomal entry or skip sites, or the use of multiple promoters. Promoter systems comprised of two separate, divergent promoters may significantly increase the size of genetic cassettes intended for use in gene therapy. However, an alternative approach is to use a single, compact, bidirectional promoter. We identified strong and stable bidirectional activity of the RPBSA synthetic promoter comprised of a fragment of the human Rpl13a promoter, together with additional intron/exon structures. The Rpl13a-based promoter drove long-term bidirectional activity of fluorescent proteins. Similar results were obtained for the EF1-α and LMP2/TAP1 promoters. However, in a lentiviral vector, the divergent bidirectional systems failed to produce sufficient titres to translate into an expression system for dual chimeric antigen receptor (CAR) expression. Although bidirectional promoters show excellent applicability to drive short RNA in Sleeping Beauty transposon systems, their possible use in the lentiviral applications requiring longer and more complex RNA, such as dual-CAR cassettes, is limited. |
| first_indexed | 2024-03-10T14:19:45Z |
| format | Article |
| id | doaj.art-bb4ddabf3661499889ad873b0c2f92ee |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-10T14:19:45Z |
| publishDate | 2020-12-01 |
| publisher | MDPI AG |
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| series | International Journal of Molecular Sciences |
| spelling | doaj.art-bb4ddabf3661499889ad873b0c2f92ee2023-11-20T23:28:40ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672020-12-012123925610.3390/ijms21239256Compact Bidirectional Promoters for Dual-Gene Expression in a Sleeping Beauty TransposonKevin He0S. M. Ali Hosseini Rad1Aarati Poudel2Alexander Donald McLellan3Department of Microbiology and Immunology, University of Otago, Dunedin 9016, New ZealandDepartment of Microbiology and Immunology, University of Otago, Dunedin 9016, New ZealandDepartment of Microbiology and Immunology, University of Otago, Dunedin 9016, New ZealandDepartment of Microbiology and Immunology, University of Otago, Dunedin 9016, New ZealandPromoter choice is an essential consideration for transgene expression in gene therapy. The expression of multiple genes requires ribosomal entry or skip sites, or the use of multiple promoters. Promoter systems comprised of two separate, divergent promoters may significantly increase the size of genetic cassettes intended for use in gene therapy. However, an alternative approach is to use a single, compact, bidirectional promoter. We identified strong and stable bidirectional activity of the RPBSA synthetic promoter comprised of a fragment of the human Rpl13a promoter, together with additional intron/exon structures. The Rpl13a-based promoter drove long-term bidirectional activity of fluorescent proteins. Similar results were obtained for the EF1-α and LMP2/TAP1 promoters. However, in a lentiviral vector, the divergent bidirectional systems failed to produce sufficient titres to translate into an expression system for dual chimeric antigen receptor (CAR) expression. Although bidirectional promoters show excellent applicability to drive short RNA in Sleeping Beauty transposon systems, their possible use in the lentiviral applications requiring longer and more complex RNA, such as dual-CAR cassettes, is limited.https://www.mdpi.com/1422-0067/21/23/9256Sleeping Beauty transposonbidirectional promotersgene expressiongene therapysynthetic biologyRPBSA |
| spellingShingle | Kevin He S. M. Ali Hosseini Rad Aarati Poudel Alexander Donald McLellan Compact Bidirectional Promoters for Dual-Gene Expression in a Sleeping Beauty Transposon International Journal of Molecular Sciences Sleeping Beauty transposon bidirectional promoters gene expression gene therapy synthetic biology RPBSA |
| title | Compact Bidirectional Promoters for Dual-Gene Expression in a Sleeping Beauty Transposon |
| title_full | Compact Bidirectional Promoters for Dual-Gene Expression in a Sleeping Beauty Transposon |
| title_fullStr | Compact Bidirectional Promoters for Dual-Gene Expression in a Sleeping Beauty Transposon |
| title_full_unstemmed | Compact Bidirectional Promoters for Dual-Gene Expression in a Sleeping Beauty Transposon |
| title_short | Compact Bidirectional Promoters for Dual-Gene Expression in a Sleeping Beauty Transposon |
| title_sort | compact bidirectional promoters for dual gene expression in a sleeping beauty transposon |
| topic | Sleeping Beauty transposon bidirectional promoters gene expression gene therapy synthetic biology RPBSA |
| url | https://www.mdpi.com/1422-0067/21/23/9256 |
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