Improve Research Reproducibility A Bio-protocol resource
bpdetail_icon_lock
Preprint

Generation of rescued mouse ES cell lines expressing EGFP-tagged Suv39h proteins

TJ Thomas Jenuwein
KS Kalina Malgorzata Swist-Rosowska

Last updated date: Mar 31, 2023 Views: 264 Forks: 0

original An abbreviated version of this protocol was published in eLife in Aug, 2017
Major satellite repeat RNA stabilize heterochromatin retention of Suv39h enzymes by RNA-nucleosome association and RNA:DNA hybrid formation
download pdf Download PDF
ask Ask a question
cite How to cite
nfavorite Favorite
  1. Sequences coding for mouse Suv39h1-EGFP, Suv39h2-EGFP and Suv39h2Δ(T3-K81)-EGFP were cloned into the pCAGS-IRES-Puro vector using the XhoI and NotI restriction sites. The plasmids were linearized using the PvuI restriction site. 
  2. 2 ml of 2% w/v gelatin (G1890, Sigma-Aldrich) in water, was add to each well of a 6-well tissue culture dish (83.3920, Sarstedt) and incubated for 20 min at room temperature.
  3. The gelatin solution was removed and 2ml of High Glucose DMEM medium (D6171, Sigma-Aldrich) containing 15% Serum Replacement (10828-028, Gibco), 2 mM L-glutamine (35050-038, Gibco), 1 mM sodium pyruvate (11360-039, Gibco), 0.1 mM beta-mercaptoethanol (21985023, Gibco), 1x non-essential amino acids (M7145, Sigma-Aldrich), 100 U/ml penicillin and 100 µg/ml streptomycin (P0781, Sigma-Aldrich),  and 1 ml of homemade Leukemia Inhibitory Factor was add to each well.
  4. Mouse Suv39h dn ES cells (d57, generated in the Jenuwein Laboratory) were plated in the gelatinized 6-well tissue culture dish at a density of 0.5x106 cells per well and allowed to recover in the tissue culture incubator.
  5. The medium was replaced with 1 ml of the fresh medium the next day in preparation for stable. transfection.
  6. 5 µg of each plasmid was diluted in Xfect Reaction Buffer (631318, Clonetech) to a final volume of 100 µl and vortexed for 5 s at the highest speed.
  7. 1.5 µl of Xfect polymer (631318, Clonetech) was added to each plasmid dilution.
  8. Samples were vortexed for 10 s at the highest speed and then incubated at room temperature for 10 min.
  9. 100 µl of the DNA-polymer solution was add to each well dropwise. 
  10. The cells were incubated for 4h at 37°C and the medium was replaced with 2ml of fresh medium.
  11. 48h after transfection, the medium was replaced with medium containing 1 μg/ml puromycin (P8833, Sigma-Aldrich). 
  12. Medium was replaced everyday with fresh medium containing 1 μg/ml puromycin, during antibiotic selection.
  13. Cells were maintained in medium containing 1 μg/ml puromycin once cell-lines were established and characterized.

 

Copyright: Content may be subjected to copyright.
How to cite：
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
  1. Jenuwein, T and Swist-Rosowska, K M(2023). Generation of rescued mouse ES cell lines expressing EGFP-tagged Suv39h proteins. Bio-protocol Preprint. bio-protocol.org/prep2186.
  2. Velazquez Camacho, O., Galan, C., Swist-Rosowska, K., Ching, R., Gamalinda, M., Karabiber, F., De La Rosa-Velazquez, I., Engist, B., Koschorz, B., Shukeir, N., Onishi-Seebacher, M., van de Nobelen, S. and Jenuwein, T.(2017). Major satellite repeat RNA stabilize heterochromatin retention of Suv39h enzymes by RNA-nucleosome association and RNA:DNA hybrid formation. eLife. DOI: 10.7554/eLife.25293

Do you have any questions about this protocol?

Post your question to gather feedback from the community. We will also invite the authors of this article to respond.

0/150

tip Tips for asking effective questions

+ Description

Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.

post Post a Question
0 Q&A
This protocol preprint was submitted via the "Request a Protocol" track.