NRAS mutant-A375 Isogenic Cell Line (ATCC^® CRL-1619IG-2^™)

BRAF is a proto-oncogene encoding B-RAF, a serine/threonine kinase of the RAF family that acts downstream of RAS and upstream of MEK in the MAPK/ERK signaling pathway. Mutations in BRAF lead to excessive cellular proliferation, differentiation, and survival. BRAF V600E mutations are present in 50% of melanomas and although there are current BRAF inhibitors used as successful therapeutics, patients often become resistant to drugs several months following treatment. One mechanism of resistance to these inhibitors is caused by a secondary NRAS Q61K acquired mutation. CRL-1619IG-2 is an isogenic cell line created at ATCC by utilizing the CRISPR/Cas9 gene editing to generate a drug resistant NRAS Q61K mutation within the A375 melanoma cell line, which naturally habors the BRAF V600E mutation. The NRASQ61K mutation in CRL-1619IG-2 has been validated at the genomic, transcript, and protein bio-functional levels. CRL-1619IG-2 shows significant resistance to the BRAF inhibitors Dabrafineb and Vemurafenib when compared to its parental cell line in 2D and 3D culture systems. CRL-1619IG-2 can be a useful model to study the RAS–RAF–MEK–ERK–MAP kinase signaling pathway and to screen potential BRAF inhibitors and anti-cancer compounds for drug discovery and development.

1. Remove and discard culture medium.
2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin- 0.53 mM EDTA solution to remove all traces of serum that contains trypsin inhibitor.
3. Add 2.0 to 3.0 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).
   Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
4. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
5. Add appropriate aliquots of the cell suspension to new culture vessels.
   Cultures can be established between 5 x 10⁴ and 1 x 10⁵ viable cells/cm².
6. Incubate cultures at 37°C.

Cormier JN, et al. Heterogeneous expression of melanoma-associated antigens and HLA-A2 in metastatic melanoma in vivo. Int. J. Cancer 75: 517-524, 1998. PubMed: 9466650

Sharma SD, et al. Melanotropic peptide-conjugated beads for microscopic visualization and characterization of melanoma melanotropin receptors. Proc. Natl. Acad. Sci. USA 93: 13715-13720, 1996. PubMed: 8943000

Gershwin ME, et al. Immunobiology of heterotransplanted human tumors in nude mice. J. Natl. Cancer Inst. 58: 1455-1463, 1977. PubMed: 857033

Giard DJ, et al. In vitro cultivation of human tumors: establishment of cell lines derived from a series of solid tumors. J. Natl. Cancer Inst. 51: 1417-1423, 1973. PubMed: 4357758

1. ATCC CRISPR/Cas9 isogenic cell lines
   

   These isogenic cell lines containing precise knock-in mutations were created by using CRISPR/Cas9 genome editing technology, followed by single cell clonal isolation and validation.

   
   Date Updated: 9/27/2017

Cormier JN, et al. Heterogeneous expression of melanoma-associated antigens and HLA-A2 in metastatic melanoma in vivo. Int. J. Cancer 75: 517-524, 1998. PubMed: 9466650

Sharma SD, et al. Melanotropic peptide-conjugated beads for microscopic visualization and characterization of melanoma melanotropin receptors. Proc. Natl. Acad. Sci. USA 93: 13715-13720, 1996. PubMed: 8943000

Gershwin ME, et al. Immunobiology of heterotransplanted human tumors in nude mice. J. Natl. Cancer Inst. 58: 1455-1463, 1977. PubMed: 857033

Giard DJ, et al. In vitro cultivation of human tumors: establishment of cell lines derived from a series of solid tumors. J. Natl. Cancer Inst. 51: 1417-1423, 1973. PubMed: 4357758