Creation of versatile cloning platforms for transgene expression and dCas9-based epigenome editing

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.

β Cell tone is defined by proglucagon peptides through cAMP signaling

“…Viral reactivation of proglucagon products. Recombinant Ad-CMV-Cre (ID HM101) and Ad-CMV-βgal (ID HD701) were generated using a new modular cloning platform, pMVP, that is described elsewhere (52). In brief, cDNA for Cre and β-gal were PCR amplified to incorporate attB4r/attB3r sites and subsequently recombined into pDONR221 P4r-P3r (Invitrogen) using BP Clonase II per the manufacturer's protocol (Invitrogen) to form MultiSite Gateway Pro entry plasmids.…”

Chromogranin B regulates early-stage insulin granule trafficking from the Golgi in pancreatic islet β-cells

“…Plasmids, viruses and stable cell line SNAP-tag was inserted near the ApaI site of human preproinsulin via gBlock synthesis (IDT) and recombined into pDONR221 P4r-P3r via BP Clonase II (Thermo Life). Rat insulin promoter (RIP)-driven proCpepSNAP-hGH polyA was assembled into a modified pAd-PL/DEST via multi-site Gateway cloning using LR Clonase II plus (Haldeman et al, 2018). Plasmid containing a U6 promoter-driven mouse-specific CgB shRNA targeting sequence or a non-targeting control (siSAFE) and a PGK promoter-expressing mCherry reporter in an adenoviral backbone was obtained from Vector Builder.…”

“…Recombinant adenoviruses were generated in HEK293 cells and purified using a cesium chloride gradient. All recombinant viruses were determined to be E1A-deficient using a quantitative PCR screen (Haldeman et al, 2018). EF1α-proCpepSNAP-hGH polyA was assembled into JN302 via multi-site Gateway cloning using LR Clonase II plus (Haldeman et al, 2018) and co-transfected with pCMV(CAT)T7-SB100 into low-passage 832/3 cells.…”

“…All recombinant viruses were determined to be E1A-deficient using a quantitative PCR screen (Haldeman et al, 2018). EF1α-proCpepSNAP-hGH polyA was assembled into JN302 via multi-site Gateway cloning using LR Clonase II plus (Haldeman et al, 2018) and co-transfected with pCMV(CAT)T7-SB100 into low-passage 832/3 cells. pCMV(CAT)T7-SB100 was Addgene plasmid #34879 (Mátés et al, 2009), deposited by Zsuzsanna Izsvak.…”

Epigenome engineering: new technologies for precision medicine

“…Precise inactivation of the enhancer's chromatin is a powerful tool to functionally characterize these tissue-specific elements for transcriptional repression activity. To this aim, SadCas9-LSD1 has been used to modify the chromatin state of a conserved enhancer, leading to altered expression of PDX1 and its target genes in insulinoma cells and in pancreatic islets ( 21 ) (Figure 2B - IV , 3A - I ).…”