KC-2060

293T-ROR2-KO-2C2-Cell-Line

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Home » 293T-ROR2-KO-2C2-Cell-Line

Background of 293T-ROR2-KO-2C2-Cell-Line

Receptor tyrosine kinase-like orphan receptor 2 (ROR2) is a receptor for the Wnt5a ligand that plays a major role during embryonic development. ROR2 expression is strongly downregulated after birth but was shown to express aberrantly in cancer as well as in other pathological conditions in the adult. In cancer, ROR2 possesses a dual role by either suppressing or promoting tumor progression in different tumor types. For example, ROR2 has a tumor suppressive function in endometrial and colon cancer, in medulloblastoma, and hepatocellular and gastric carcinoma. In several other tumor types including cancers from the breast, prostate, and ovary among others, ROR2 promotes cancer. The underlying reasons for this discrepancy have not been elucidated and ROR2 has been considered alternatively an oncogene or a tumor suppressor gene depending on the tumor type. Interestingly, ROR2 exerts these dual functions by regulating the same biological processes in opposite ways. For instance, the anti- and pro-tumoral function of ROR2 have been associated with the inhibition or stimulation of cell proliferation, respectively. Similarly, ROR2 was shown to either enhance or inhibit cell migration and invasion.

Specifications

Catalog NumberKC-2060
Cell Line Name293T-ROR2-KO-2C2-Cell-Line
Host Cell Line293T
DescriptionStable 293T clone with human ROR2 gene knockout, No.2C2
QuantityTwo vials of frozen cells (≥2-106/vial)
StabilityStable in culture over a minimum of 10 passages
ApplicationDrug screening and biological assays
Freezing MediumDMEM+20% FBS+10% DMSO
Propagation MediumDMEM+10% FBS
Selection MarkerN/A
Morphologyepithelial
SubcultureSplit saturated culture 1:5-1:8 every 2-3 days; seed out at about 1-3 × 105 cells/mL
Incubation37 °C with 5% CO2
StorageLiquid nitrogen immediately upon receiving
Doubling TimeApproximately 30 hours
Mycoplasma StatusNegative
In Vivo ValidationNA

Cell Line Generation

293T-ROR2-KO-2C2 cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of 293T-ROR2-KO-2C2 cell line stable clone using PCR sequencing.

Figure 2: Characterization of 293T-ROR2-KO-2C2 cell line stable clone using RT-PCR sequencing.

Figure 3: Characterization of 293T-ROR2-KO-2C2 cell line stable clone using FACS.

Cell Resuscitation

  1. Prewarm culture medium (DMEM + 10% FBS)in a 37°C water bath.
  2. Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
  3. Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
  4. Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
  5. Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
  6. Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
  7. Incubate the flask at 37°C, 5% CO2 incubator.
  8. Split saturated culture 1:5-1:8 every 2-3 days; seed out at about 1-3 × 105 cells/mL.

Cell Freezing

  1. Prepare the freezing medium (70% DMEM + 20% FBS + 10% DMSO) fresh immediately before use.
  2. Keep the freezing medium on ice and label cryovials.
  3. Transfer cells to a sterile, conical centrifuge tube, and count the cells.
  4. Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
  5. Resuspend the cells at a density of at least 3×106 cells/mL in chilled freezing medium.
  6. Aliquot 1 mL of the cell suspension into each cryovial.
  7. Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
  8. Transfer vials to liquid nitrogen for long-term storage

References

  1. Lopez-Bergami P. ROR2, a driver of phenotype switching in melanoma? Cancer Cell Int. 2022 Sep 20;22(1):288. doi: 10.1186/s12935-022-02711-x. PMID: 36127680; PMCID: PMC9487041.
  2. Castro MV, Lopez-Bergami P. Cellular and molecular mechanisms implicated in the dual role of ROR2 in cancer. Crit Rev Oncol Hematol. 2022 Feb;170:103595. doi: 10.1016/j.critrevonc.2022.103595. Epub 2022 Jan 13. PMID: 35032666.
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