Abstract
Genetic manipulation via transgene overexpression, RNAi, or Cas9-based methods is central to biomedical research. Unfortunately, use of these tools is often limited by vector options. We have created a modular platform (pMVP) that allows a gene of interest to be studied in the context of an array of promoters, epitope tags, conditional expression modalities, and fluorescent reporters, packaged in 35 custom destination vectors, including adenovirus, lentivirus, PiggyBac transposon, and Sleeping Beauty transposon, in aggregate >108,000 vector permutations. We also used pMVP to build an epigenetic engineering platform, pMAGIC, that packages multiple gRNAs and either Sa-dCas9 or x-dCas9(3.7) fused to one of five epigenetic modifiers. Importantly, via its compatibility with adenoviral vectors, pMAGIC uniquely enables use of dCas9/LSD1 fusions to interrogate enhancers within primary cells. To demonstrate this, we used pMAGIC to target Sa-dCas9/LSD1 and modify the epigenetic status of a conserved enhancer, resulting in altered expression of the homeobox transcription factor PDX1 and its target genes in pancreatic islets and insulinoma cells. In sum, the pMVP and pMAGIC systems empower researchers to rapidly generate purpose-built, customized vectors for manipulation of gene expression, including via targeted epigenetic modification of regulatory elements in a broad range of disease-relevant cell types.
Highlights
A core strategy for biomedical research is to genetically manipulate specific components of complex physiological systems to define regulatory mechanisms and disease-causing pathways
Using pMAGIC-derived Ad5 vectors, we demonstrate that Sa-dead Cas9 (dCas9)/LSD1-mediated epigenetic modification of the Area IV element causes a significant inhibition of endogenous PDX1 expression in both the INS1 832/13 rat insulinoma cell line and in primary rat pancreatic islets
High-fidelity assembly of unique vectors in a user-friendly manner, we utilized Multisite Gateway® Pro technology [12] as a foundation to generate a suite of entry plasmids and promoterless Gateway destination vectors that we term globally as ‘plasmid-based modular vector platform (pMVP)’, through which a gene of interest can be paired with combinations of promoter/polyadenylation signal (Figures 1A, 2A, 3A) or promoter/N-terminal fusion/polyA (Figures 1A, 2B and 3B) elements and recombined into a pDEST vector (Figures 1B, 2A and B) via an overnight recombination reaction
Summary
A core strategy for biomedical research is to genetically manipulate specific components of complex physiological systems to define regulatory mechanisms and disease-causing pathways Such strategies can be hampered by limitations imposed by current systems for delivery of transgenes or gene suppressors into specific cell-types central to disease etiology. As experimental questions evolve throughout the course of a project, it is frequently desirable to utilize other experimental models (e.g. stable cell lines, transient expression) to obtain mechanistic insight. This pivot to a new model is often hampered by the lack of cross-vector compatibility. We describe innovative modular cloning platforms that enable creation of highly customized adenovirus, expression plasmid, lentivirus, PiggyBac (PB) transposon or Sleeping Beauty (SB) transposon vectors for transgene or RNAi delivery, as well as dCas9-mediated epigenetic engineering vectors, that allow deployment of a cDNA, shRNA or epigenome editing modality in a customized gene delivery vector in three to 5 days
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