Construction of the PUF RNA-binding Domain Scaffold with Customized RNA Binding Specificity by Overlapping PCR
2:05
Transfection of Engineered Splicing Factors (ESFs) for Modulation of Exon Inclusion and Alternative Splice-site Use
3:43
Cell Harvest and Identification of Spliced Isoforms
6:24
Modulation of Endogenous Bcl-x Splicing and Apoptosis by ESFs
8:23
Results: Subcellular Localization of ESFs in Transfected HeLa Cells
9:30
Conclusion
Transcription
The overall goal of this protocol is to engineer artificial splicing factors that can specifically manipulate alternative splicing of a given target. This method can help to answer key questions in the field of RNA processing, such as the regulati
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This report describes a bioengineering method to design and construct novel Artificial Splicing Factors (ASFs) that specifically modulate the splicing of target genes in mammalian cells. This method can be further expanded to engineer various artificial factors to manipulate other aspects of RNA metabolism.