Cas9 Expressing RCS Cell Line
|T3290||1x106 cells / 1.0 ml|
|Description||Swarm rat chondrosarcoma (RCS) cells were derived from neoplasm in rat spine and is a model for chondrosarcoma tumor development. The Cas9 expressing RCS Cell Line was established through co-transfection with hCas9_P2A_mCherry_T2a_Puro and pOG44 vector. The generation of the Cas9 expressing RCS cell line allows users to generate targeted mutations in genes via transfection or lentiviral transfection with single-guide RNAs (sgRNAs). This cell line is a valuable research tool which enables users endless possibilities to design their desired cell line for the study of chondrocytes and gene function.|
|Cas9 Expression||Stably Integrated Cas9|
|Unit quantity||1x106 cells / 1.0 ml|
|Growth Properties||Adherent, Suspension|
|Seeding Density||Thaw entire contents into an appropriate T25 flask as specified in the Propagation instructions.|
|Freeze-Thaw Recovery||1. Pre-warm complete media in a 37°C waterbath.|
2. Remove the cryopreserved vial from the liquid nitrogen storage tank.
3. Thaw the cells quickly by placing the lower half of the vial into the 37°C water bath and remove after 60 seconds. There should still be a few ice crystals left after thawing. It is important not to over-thaw the cryovials as the presence of DMSO is toxic to the cells.
4. Re-suspend the cells in the vial and transfer the cell suspension into a 15mL sterile conical tube containing 5mL complete media.
5. Centrifuge cells at 1500rpm for 3 minutes to pellet.
6. Aspirate out the media, leaving cell pellet undisturbed.
7. Re-suspend pellet in fresh culture medium and plate in new culture vessel.
8. Incubate cultures at 37°C, 5% CO2.
|Shipping Conditions||Dry Ice|
|Quality Control||Western blot and qRT-PCR to show Cas9 transgene expression|
- Important Considerations for Cells
- Cell Handling Instructions Upon Arrival
- Subculturing Protocol
- Freezing Protocol
- Thawing Protocol
|What is the PAM sequence required for sgRNA design for use with these cells?|
We use spCas9, therefore the PAM is 5'-NGG-3' for this cell line.
|Is there a selection marker for cas9 expression?|
Yes, Puromycin is selection marker for most of these cell lines. Some cell lines may have additional inherent drug resistance eg. 293T has neomycin resistance. Please refer to the product page for the specific cell line of interest.
- Yang, M et al. "CRISPR/Cas9 mediated generation of stable chondrocyte cell lines with targeted gene knockouts; analysis of an aggrecan knockout cell line" Bone 69:118-125 (2014). DOI: 10.1016/j.bone.2014.09.005.