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

Publication ,  Journal Article
Haldeman, JM; Conway, AE; Arlotto, ME; Slentz, DH; Muoio, DM; Becker, TC; Newgard, CB
Published in: Nucleic Acids Res
February 28, 2019

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.

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Published In

Nucleic Acids Res

DOI

EISSN

1362-4962

Publication Date

February 28, 2019

Volume

47

Issue

4

Start / End Page

e23

Location

England

Related Subject Headings

  • Transgenes
  • Trans-Activators
  • Rats
  • Promoter Regions, Genetic
  • Mice
  • Lentivirus
  • Islets of Langerhans
  • Insulinoma
  • Humans
  • Homeodomain Proteins
 

Citation

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Haldeman, J. M., Conway, A. E., Arlotto, M. E., Slentz, D. H., Muoio, D. M., Becker, T. C., & Newgard, C. B. (2019). Creation of versatile cloning platforms for transgene expression and dCas9-based epigenome editing. Nucleic Acids Res, 47(4), e23. https://doi.org/10.1093/nar/gky1286
Haldeman, Jonathan M., Amanda E. Conway, Michelle E. Arlotto, Dorothy H. Slentz, Deborah M. Muoio, Thomas C. Becker, and Christopher B. Newgard. “Creation of versatile cloning platforms for transgene expression and dCas9-based epigenome editing.Nucleic Acids Res 47, no. 4 (February 28, 2019): e23. https://doi.org/10.1093/nar/gky1286.
Haldeman JM, Conway AE, Arlotto ME, Slentz DH, Muoio DM, Becker TC, et al. Creation of versatile cloning platforms for transgene expression and dCas9-based epigenome editing. Nucleic Acids Res. 2019 Feb 28;47(4):e23.
Haldeman, Jonathan M., et al. “Creation of versatile cloning platforms for transgene expression and dCas9-based epigenome editing.Nucleic Acids Res, vol. 47, no. 4, Feb. 2019, p. e23. Pubmed, doi:10.1093/nar/gky1286.
Haldeman JM, Conway AE, Arlotto ME, Slentz DH, Muoio DM, Becker TC, Newgard CB. Creation of versatile cloning platforms for transgene expression and dCas9-based epigenome editing. Nucleic Acids Res. 2019 Feb 28;47(4):e23.
Journal cover image

Published In

Nucleic Acids Res

DOI

EISSN

1362-4962

Publication Date

February 28, 2019

Volume

47

Issue

4

Start / End Page

e23

Location

England

Related Subject Headings

  • Transgenes
  • Trans-Activators
  • Rats
  • Promoter Regions, Genetic
  • Mice
  • Lentivirus
  • Islets of Langerhans
  • Insulinoma
  • Humans
  • Homeodomain Proteins