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Why endogenous tagging?​

​Image with unprecedented clarity compared to other tagging methods​
 

Endogenous tagging of intracellular structures with gene editing​

A. α-tubulin transfection
​​α​-tubulin gene-edited
B. Tom20 transfection
​Tom20 gene-edited
C. Desmoplakin transfection
​Desmoplakin gene-edited
Figure. Gene-editing versus transient transfection. Z-stacks of live hiPS cells comparing endogenous mEGFP tagging via gene-editing versus overexpression via transient transfection. All movies were imaged with a laser-scanning confocal microscope and images start from the bottom of the cells and end at the top. Movies shows the transient transfections and right panels the gene-edited cell lines for fluorescently tagged α-tubulin (A), desmoplakin (B) and Tom20 (C).​
Observations
Labeling structures with mEGFP endogenously (via CRISPR/cas9 gene editing) generates human cells that we can image with unprecedented clarity compared to tagged proteins that are generated by transient transfection. The extent of the difference can vary:
  • Tagged α-tubulin: microtubules are more clearly visible due to decreased background when compared to overexpression
  • Tagged desmomplakin: desmosomes localize to small puncta near the tops of cells. Transiently transfected cells form more and larger desmosomes or artificial desmosome-like structures.
  • Tagged Tom20: overexpression of tagged Tom20 often generates sick cells with completely misshapen mitochondria compared to tagged Tom20 expressed at endogenous levels.

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  • About
      Institute
      1. News feed
      2. What we do
      3. Publications
      4. Allen Institute | allenInstitute.org
      5. Careers
      Site
      1. Home page
      2. Site updates
      3. Archived content
  • Allen Cell Collection
      Order cells & plasmids
      1. Cell Catalog
      2. Cell Catalog quickview
      3. Cell Shorts (documentaries on labs using our cells)
      4. Support forum
      Lab methods
      1. Instructional videos for success in the lab
      2. Standard operating procedures (written methods)
      3. Illustrated overviews
      About our hiPS cells
      1. hiPS Cell Structure Overview
      2. Visual Guide to Human Cells
      3. Cell structure observations
      4. Why endogenous tagging?
      5. Differentiation into cardiomyocytes
      6. Genomics
      7. Download cell data (images, genomics, features)
  • Data & Digital Tools
      Online image analysis
      1. Cell Feature Explorer (plotting & 3D viewer)
      2. 3D cell viewer (pre2018)
      3. Deep cell zoom (216,016 cells)
      Online modeling viewers
      1. Visual Guide to Human Cells
      2. Simularium (4D visual analysis)
      3. Integrated Mitotic Stem Cell
      4. └ Z-stack viewer
      5. └ 3D viewer
      6. Allen Integrated Cell viewer
      7. Label-free examples viewer
      8. 3D probabilistic model viewer
      Desktop tools
      1. Allen Cell & Structure Segmenter
      2. AGAVE 3D pathtrace image viewer
      Data & code
      1. Download cell data (images, genomics, features)
      2. Code repositories & software
  • Analysis & Modeling
      Allen Integrated Cell models
      1. Overview
      2. Integrated Mitotic Stem Cell
      3. └ Z-stack viewer
      4. └ 3D viewer
      5. Label-free Determination
      6. └ 3D viewer
      7. 3D Probabilistic Modeling
      8. └ 3D viewer
      9. Visual Guide to Human Cells
      4D biology models
      1. Simularium (online 4D viewer)
      Methodologies
      1. Drug perturbation pilot study
      2. hiPS cells during mitosis
      3. Differentiation into cardiomyocytes
  • Publications
      Articles
      1. All journal publications
      2. Preprints (biorxiv, arxiv)
      Posters
      1. Select posters
  • Education
      Education resources
      1. All Resources
      2. Teaching materials
      Online tools popular with teachers
      1. Visual Guide to Human Cells
      2. Integrated Mitotic Stem Cell
      3. Cell Feature Explorer (interactive plotting & 3D viewer)
      4. 3D cell viewer (pre2018 data)
      5. hiPS cell structure overview
  • Support
      Questions
      1. FAQs
      2. Forum
      Tutorials for digital tools
      1. Digital tool tutorials with videos
      2. Visual Guide tutorial
      3. AGAVE user guide
      Lab methods
      1. Instructional videos for success in the lab
      2. Standard operating procedures (written methods)
      3. Illustrated overviews
  • 🔍
      SEARCHBAR