A novel retroviral vector system to analyze expression from mRNA with retained introns using fluorescent proteins and flow cytometry
| Autor: | Marie-Louise Hammarskjold, David Rekosh, M. Sharma, Patrick E. H. Jackson, Jing Huang, Sara K. Rasmussen |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2019 |
| Předmět: |
0301 basic medicine
Gene isoform Viral protein Genetic Vectors Green Fluorescent Proteins lcsh:Medicine Biology medicine.disease_cause Cell Line Viral vector 03 medical and health sciences 0302 clinical medicine Transduction Genetic medicine Humans RNA Messenger lcsh:Science Gene Messenger RNA Reporter gene Multidisciplinary lcsh:R Intron RNA Flow Cytometry Introns Cell biology 030104 developmental biology Gene Expression Regulation HIV-1 lcsh:Q 030217 neurology & neurosurgery |
| Zdroj: | Scientific Reports, Vol 9, Iss 1, Pp 1-14 (2019) |
| ISSN: | 2045-2322 |
| DOI: | 10.1038/s41598-019-42914-3 |
| Popis: | The ability to overcome cellular restrictions that exist for the export and translation of mRNAs with retained introns is a requirement for the replication of retroviruses and also for the expression of many mRNA isoforms transcribed from cellular genes. In some cases, RNA structures have been identified in the mRNA that directly interact with cellular factors to promote the export and expression of isoforms with retained introns. In other cases, a viral protein is also required to act as an adapter. In this report we describe a novel vector system that allows measurement of the ability of cis- and trans-acting factors to promote the export and translation of mRNAs with retained introns. One reporter vector used in this system is derived from an HIV proviral clone engineered to express two different fluorescent proteins from spliced and unspliced transcripts. The ratio of fluorescent signals is a measurement of the efficiency of export and translation. A second vector utilizes a third fluorescent protein to measure the expression of viral export proteins that interact with some of the export elements. Both vectors can be packaged into viral particles and be used to transduce cells, allowing expression at physiological levels from the integrated vector. |
| Databáze: | OpenAIRE |
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