KC-4798

293T-ROR1 Cell Line

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Background of 293T-ROR1 Cell Line

ROR1 (Receptor Tyrosine Kinase Like Orphan Receptor 1) is a Protein Coding gene.This gene encodes a receptor tyrosine kinase-like orphan receptor that modulates neurite growth in the central nervous system. The encoded protein is a glycosylated type I membrane protein that belongs to the ROR subfamily of cell surface receptors. It is a pseudokinase that lacks catalytic activity and may interact with the non-canonical Wnt signalling pathway. This gene is highly expressed during early embryonic development but expressed at very low levels in adult tissues. Increased expression of this gene is associated with B-cell chronic lymphocytic leukaemia. Alternative splicing results in multiple transcript variants encoding different isoforms.

Specifications

Catalog Number	KC-4798
Cell Line Name	293T-ROR1 Cell Line
Host Cell Line	293T
Description	293T cell line stably expressing exogenous ROR1 gene
Quantity	Two vials of frozen cells (≥2-10⁶/vial)
Stability	Stable in culture over a minimum of 10 passages
Application	Drug screening and biological assays
Freezing Medium	70% DMEM + 20% FBS + 10% DMSO
Propagation Medium	DMEM + 10% FBS + 1 μg/mL Puromycin
Selection Marker	Puromycin
Morphology	Fibroblastoid cells growing as monolayer
Subculture	Split saturated culture 1:4-1:5 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL
Incubation	37 °C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Approximately 30 hours
Mycoplasma Status	Negative

Cell Line Generation

293T-ROR1 cell line was generated using lentiviral vector expressing ROR1 sequence.

Characterization

Figure1: Characterization of mouse ROR1 overexpressing in 293T stable clones using FACS.

Figure2: Characterization of 293T-ROR1 cell line stable clone using PCR sequencing.

Cell Resuscitation

1. Prewarm culture medium (DMEM supplemented with 10% FBS and 1 μg/mL puromycin)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 1:4-1:5 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. Zhang S, Cui B, Lai H, Liu G, Ghia EM, Widhopf GF 2nd, Zhang Z, Wu CC, Chen L, Wu R, Schwab R, Carson DA, Kipps TJ. Ovarian cancer stem cells express ROR1, which can be targeted for anti-cancer-stem-cell therapy. Proc Natl Acad Sci U S A. 2014 Dec 2;111(48):17266-71. doi: 10.1073/pnas.1419599111. Epub 2014 Nov 19. PMID: 25411317; PMCID: PMC4260559.
2. Liu D, Gunther K, Enriquez LA, Daniels B, O'Mara TA, Tang K, Spurdle AB, Ford CE. ROR1 is upregulated in endometrial cancer and represents a novel therapeutic target. Sci Rep. 2020 Aug 17;10(1):13906. doi: 10.1038/s41598-020-70924-z. Erratum in: Sci Rep. 2022 Jun 28;12(1):10918. doi: 10.1038/s41598-022-15043-7. PMID: 32807831; PMCID: PMC7431863.