Principles and Practical Considerations for the Analysis of Disease-Associated Alternative Splicing Events Using the Gateway Cloning-Based Minigene Vectors pDESTsplice and pSpliceExpress
Splicing is an important RNA processing step. Genetic variations can alter the splicing process and thereby contribute to the development of various diseases. Alterations of the splicing pattern can be examined by gene expression analyses, by computational tools for predicting the effects of genetic...
| Main Authors: | , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2021-05-01
|
| Series: | International Journal of Molecular Sciences |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1422-0067/22/10/5154 |
| _version_ | 1797534344198225920 |
|---|---|
| author | Elena Putscher Michael Hecker Brit Fitzner Peter Lorenz Uwe Klaus Zettl |
| author_facet | Elena Putscher Michael Hecker Brit Fitzner Peter Lorenz Uwe Klaus Zettl |
| author_sort | Elena Putscher |
| collection | DOAJ |
| description | Splicing is an important RNA processing step. Genetic variations can alter the splicing process and thereby contribute to the development of various diseases. Alterations of the splicing pattern can be examined by gene expression analyses, by computational tools for predicting the effects of genetic variants on splicing, and by splicing reporter minigene assays for studying alternative splicing events under defined conditions. The minigene assay is based on transient transfection of cells with a vector containing a genomic region of interest cloned between two constitutive exons. Cloning can be accomplished by the use of restriction enzymes or by site-specific recombination using Gateway cloning. The vectors pDESTsplice and pSpliceExpress represent two minigene systems based on Gateway cloning, which are available through the Addgene plasmid repository. In this review, we describe the features of these two splicing reporter minigene systems. Moreover, we provide an overview of studies in which determinants of alternative splicing were investigated by using pDESTsplice or pSpliceExpress. The studies were reviewed with regard to the investigated splicing regulatory events and the experimental strategy to construct and perform a splicing reporter minigene assay. We further elaborate on how analyses on the regulation of RNA splicing offer promising prospects for gaining important insights into disease mechanisms. |
| first_indexed | 2024-03-10T11:28:19Z |
| format | Article |
| id | doaj.art-f751d49b69c04e339f7fff9951b00b06 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-10T11:28:19Z |
| publishDate | 2021-05-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-f751d49b69c04e339f7fff9951b00b062023-11-21T19:30:25ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672021-05-012210515410.3390/ijms22105154Principles and Practical Considerations for the Analysis of Disease-Associated Alternative Splicing Events Using the Gateway Cloning-Based Minigene Vectors pDESTsplice and pSpliceExpressElena Putscher0Michael Hecker1Brit Fitzner2Peter Lorenz3Uwe Klaus Zettl4Division of Neuroimmunology, Department of Neurology, Rostock University Medical Center, Gehlsheimer Street 20, 18147 Rostock, GermanyDivision of Neuroimmunology, Department of Neurology, Rostock University Medical Center, Gehlsheimer Street 20, 18147 Rostock, GermanyDivision of Neuroimmunology, Department of Neurology, Rostock University Medical Center, Gehlsheimer Street 20, 18147 Rostock, GermanyRostock University Medical Center, Institute of Immunology, Schillingallee 70, 18057 Rostock, GermanyDivision of Neuroimmunology, Department of Neurology, Rostock University Medical Center, Gehlsheimer Street 20, 18147 Rostock, GermanySplicing is an important RNA processing step. Genetic variations can alter the splicing process and thereby contribute to the development of various diseases. Alterations of the splicing pattern can be examined by gene expression analyses, by computational tools for predicting the effects of genetic variants on splicing, and by splicing reporter minigene assays for studying alternative splicing events under defined conditions. The minigene assay is based on transient transfection of cells with a vector containing a genomic region of interest cloned between two constitutive exons. Cloning can be accomplished by the use of restriction enzymes or by site-specific recombination using Gateway cloning. The vectors pDESTsplice and pSpliceExpress represent two minigene systems based on Gateway cloning, which are available through the Addgene plasmid repository. In this review, we describe the features of these two splicing reporter minigene systems. Moreover, we provide an overview of studies in which determinants of alternative splicing were investigated by using pDESTsplice or pSpliceExpress. The studies were reviewed with regard to the investigated splicing regulatory events and the experimental strategy to construct and perform a splicing reporter minigene assay. We further elaborate on how analyses on the regulation of RNA splicing offer promising prospects for gaining important insights into disease mechanisms.https://www.mdpi.com/1422-0067/22/10/5154alternative splicingRNA processingsplicing regulationsplicing reporter minigene assaygateway cloningpDESTsplice |
| spellingShingle | Elena Putscher Michael Hecker Brit Fitzner Peter Lorenz Uwe Klaus Zettl Principles and Practical Considerations for the Analysis of Disease-Associated Alternative Splicing Events Using the Gateway Cloning-Based Minigene Vectors pDESTsplice and pSpliceExpress International Journal of Molecular Sciences alternative splicing RNA processing splicing regulation splicing reporter minigene assay gateway cloning pDESTsplice |
| title | Principles and Practical Considerations for the Analysis of Disease-Associated Alternative Splicing Events Using the Gateway Cloning-Based Minigene Vectors pDESTsplice and pSpliceExpress |
| title_full | Principles and Practical Considerations for the Analysis of Disease-Associated Alternative Splicing Events Using the Gateway Cloning-Based Minigene Vectors pDESTsplice and pSpliceExpress |
| title_fullStr | Principles and Practical Considerations for the Analysis of Disease-Associated Alternative Splicing Events Using the Gateway Cloning-Based Minigene Vectors pDESTsplice and pSpliceExpress |
| title_full_unstemmed | Principles and Practical Considerations for the Analysis of Disease-Associated Alternative Splicing Events Using the Gateway Cloning-Based Minigene Vectors pDESTsplice and pSpliceExpress |
| title_short | Principles and Practical Considerations for the Analysis of Disease-Associated Alternative Splicing Events Using the Gateway Cloning-Based Minigene Vectors pDESTsplice and pSpliceExpress |
| title_sort | principles and practical considerations for the analysis of disease associated alternative splicing events using the gateway cloning based minigene vectors pdestsplice and pspliceexpress |
| topic | alternative splicing RNA processing splicing regulation splicing reporter minigene assay gateway cloning pDESTsplice |
| url | https://www.mdpi.com/1422-0067/22/10/5154 |
| work_keys_str_mv | AT elenaputscher principlesandpracticalconsiderationsfortheanalysisofdiseaseassociatedalternativesplicingeventsusingthegatewaycloningbasedminigenevectorspdestspliceandpspliceexpress AT michaelhecker principlesandpracticalconsiderationsfortheanalysisofdiseaseassociatedalternativesplicingeventsusingthegatewaycloningbasedminigenevectorspdestspliceandpspliceexpress AT britfitzner principlesandpracticalconsiderationsfortheanalysisofdiseaseassociatedalternativesplicingeventsusingthegatewaycloningbasedminigenevectorspdestspliceandpspliceexpress AT peterlorenz principlesandpracticalconsiderationsfortheanalysisofdiseaseassociatedalternativesplicingeventsusingthegatewaycloningbasedminigenevectorspdestspliceandpspliceexpress AT uweklauszettl principlesandpracticalconsiderationsfortheanalysisofdiseaseassociatedalternativesplicingeventsusingthegatewaycloningbasedminigenevectorspdestspliceandpspliceexpress |