KC-4375

COLO205-REV3L-KO Cell Line

52715

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Background of COLO205-REV3L-KO Cell Line

The human Rev3L gene encodes a ~350 kDa protein (REV3L) containing a large C-terminal DNA polymerase subunit. Human Rev3L is ubiquitously expressed in tissues and is involved in the bypass of many types of DNA damage induced by ultraviolet (UV) radiation and chemical damaging agents. Deletion of Rev3 is embryonically lethal, whereas over-expression of Rev3 leads to increased spontaneous mutations. The encoded protein can be found in mitochondria, where it protects DNA from damage. Defects in this gene are a cause of Mobius syndrome.

Specifications

Catalog Number	KC-4375
Cell Line Name	COLO205-REV3L-KO Cell Line
Host Cell Line	COLO205
Description	Stable COLO205 clone with human REV3L gene knockout, No.3B2
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	RPMI1640+20% FBS+10% DMSO
Propagation Medium	RPMI1640+10% FBS
Selection Marker	NA
Morphology	epithelial
Subculture	Split saturated culture 1:5-1:8 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 27 hours
Mycoplasma Status	Negative

Cell Line Generation

COLO205-REV3L-KO-3B2 cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of COLO205-REV3L-KO-3B2 cell line stable clone using PCR sequencing.

Figure 2: Characterization of COLO205-REV3L-KO-3B2 cell line stable clone using RT-PCR sequencing.

Cell Resuscitation

Prewarm culture medium (RPMI1640 + 10% FBS) in a 37°C water bath.
Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
Incubate the flask at 37°C, 5% CO₂ incubator.
Split saturated culture 1:10 every 3 days; seed out at about 1-3 ×10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% RPMI1640 + 20% FBS + 10% DMSO) fresh immediately before use.
Keep the freezing medium on ice and label cryovials.
Transfer cells to a sterile, conical centrifuge tube, and count the cells.
Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
Resuspend the cells at a density of at least 3×10⁶ cells/mL in chilled freezing medium.
Aliquot 1 mL of the cell suspension into each cryovial.
Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
Transfer vials to liquid nitrogen for long-term storage.

References

Singh B, Li X, Owens KM, Vanniarajan A, Liang P, Singh KK. Human REV3 DNA Polymerase Zeta Localizes to Mitochondria and Protects the Mitochondrial Genome. PLoS One. 2015 Oct 13;10(10):e0140409. doi: 10.1371/journal.pone.0140409. PMID: 26462070; PMCID: PMC4604079.
Tomas-Roca L, Tsaalbi-Shtylik A, Jansen JG, Singh MK, Epstein JA, Altunoglu U, Verzijl H, Soria L, van Beusekom E, Roscioli T, Iqbal Z, Gilissen C, Hoischen A, de Brouwer APM, Erasmus C, Schubert D, Brunner H, Pérez Aytés A, Marin F, Aroca P, Kayserili H, Carta A, de Wind N, Padberg GW, van Bokhoven H. De novo mutations in PLXND1 and REV3L cause Möbius syndrome. Nat Commun. 2015 Jun 12;6:7199. doi: 10.1038/ncomms8199. PMID: 26068067; PMCID: PMC4648025.