Skip to main content
bioRxiv
  • Home
  • About
  • Submit
  • ALERTS / RSS
Advanced Search
New Results

Scarless gene tagging of transcriptionally silent genes in hiPSCs to visualize cardiomyocyte sarcomeres in live cells

Brock Roberts, Joy Arakaki, Kaytlyn A. Gerbin, Haseeb Malik, Angelique Nelson, Melissa C. Hendershott, Caroline Hookway, Susan A. Ludmann, Irina A. Mueller, Ruian Yang, Susanne M. Rafelski, Ruwanthi N. Gunawardane
doi: https://doi.org/10.1101/342881
Brock Roberts
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Joy Arakaki
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Kaytlyn A. Gerbin
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Haseeb Malik
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Angelique Nelson
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Melissa C. Hendershott
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Caroline Hookway
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Susan A. Ludmann
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Irina A. Mueller
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Ruian Yang
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Susanne M. Rafelski
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Ruwanthi N. Gunawardane
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • Abstract
  • Full Text
  • Info/History
  • Metrics
  • Preview PDF
Loading

Abstract

We describe a multi-step CRISPR/Cas9 gene editing method to create endogenously tagged GFP-fusions of transcriptionally silent genes in human induced pluripotent stem cells (hiPSCs), allowing visualization of proteins that are only expressed upon differentiation. To do this, we designed a donor template containing the monomeric EGFP (mEGFP) fusion tag and an mCherry selection cassette delivered in tandem to a target locus via homology directed repair (HDR). mCherry expression was driven by a constitutive promoter and served as a drug-free, excisable selection marker. Following selection, the mCherry cassette was excised with Cas9, creating an mEGFP-fusion with the target gene. We achieved scarless excision by using repetitive sequences to guide microhomology-mediated end joining (MMEJ) and introduce linker sequences between the mEGFP tag and the target gene. Using this strategy, we successfully tagged genes encoding the cardiomyocyte sarcomeric proteins troponin I (TNNI1), alpha-actinin (ACTN2), titin (TTN), myosin light chain 2a (MYL7), and myosin light chain 2v (MYL2) with mEGFP in undifferentiated hiPSCs. This methodology provides a general strategy for scarlessly introducing tags to transcriptionally silent loci in hiPSCs.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
Back to top
PreviousNext
Posted June 09, 2018.
Download PDF
Email

Thank you for your interest in spreading the word about bioRxiv.

NOTE: Your email address is requested solely to identify you as the sender of this article.

Enter multiple addresses on separate lines or separate them with commas.
Scarless gene tagging of transcriptionally silent genes in hiPSCs to visualize cardiomyocyte sarcomeres in live cells
(Your Name) has forwarded a page to you from bioRxiv
(Your Name) thought you would like to see this page from the bioRxiv website.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
Scarless gene tagging of transcriptionally silent genes in hiPSCs to visualize cardiomyocyte sarcomeres in live cells
Brock Roberts, Joy Arakaki, Kaytlyn A. Gerbin, Haseeb Malik, Angelique Nelson, Melissa C. Hendershott, Caroline Hookway, Susan A. Ludmann, Irina A. Mueller, Ruian Yang, Susanne M. Rafelski, Ruwanthi N. Gunawardane
bioRxiv 342881; doi: https://doi.org/10.1101/342881
Digg logo Reddit logo Twitter logo Facebook logo Google logo LinkedIn logo Mendeley logo
Citation Tools
Scarless gene tagging of transcriptionally silent genes in hiPSCs to visualize cardiomyocyte sarcomeres in live cells
Brock Roberts, Joy Arakaki, Kaytlyn A. Gerbin, Haseeb Malik, Angelique Nelson, Melissa C. Hendershott, Caroline Hookway, Susan A. Ludmann, Irina A. Mueller, Ruian Yang, Susanne M. Rafelski, Ruwanthi N. Gunawardane
bioRxiv 342881; doi: https://doi.org/10.1101/342881

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
  • Tweet Widget
  • Facebook Like
  • Google Plus One

Subject Area

  • Cell Biology
Subject Areas
All Articles
  • Animal Behavior and Cognition (4095)
  • Biochemistry (8786)
  • Bioengineering (6493)
  • Bioinformatics (23386)
  • Biophysics (11766)
  • Cancer Biology (9167)
  • Cell Biology (13290)
  • Clinical Trials (138)
  • Developmental Biology (7422)
  • Ecology (11386)
  • Epidemiology (2066)
  • Evolutionary Biology (15119)
  • Genetics (10413)
  • Genomics (14024)
  • Immunology (9145)
  • Microbiology (22108)
  • Molecular Biology (8793)
  • Neuroscience (47445)
  • Paleontology (350)
  • Pathology (1423)
  • Pharmacology and Toxicology (2483)
  • Physiology (3711)
  • Plant Biology (8063)
  • Scientific Communication and Education (1433)
  • Synthetic Biology (2215)
  • Systems Biology (6021)
  • Zoology (1251)