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

Organism: Homo sapiens, human  /  Tissue: skin  /  Disease: malignant melanoma

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Organism Homo sapiens, human
Tissue skin
Product Format frozen 1.0 mL
Morphology epithelial
Culture Properties adherent
Biosafety Level 2

Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.

Disease malignant melanoma
Age 54 years
Gender female
Applications

NRASQ61K; BRAFV600E research, anti-cancer drug screening, BRAF drug resistant melanoma model, RAS-RAF kinase signaling pathway. Ideally the parental cell line (ATCC® CRL-1619™) is included as a control for drug-sensitive response in experiments with this cell line.

Images Cell Micrograph of NRAS mutant-A375 Isogenic Cell Line, ATCC CRL-1619IG-2 NRAS Mutant A-375 Isogenic Cells CRL-1619IG2
Comments

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.

Complete Growth Medium The base medium for this cell line is ATCC-formulated Dulbecco’s Modified Eagle’s Medium (DMEM; ATCC® 30-2002™). To make the complete medium add the following component to the base medium: Fetal bovine serum (FBS; ATCC® 30-2020™) to a final concentration of 10%.
Subculturing
Volumes used in this protocol are for 75 cm2 flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes. Corning® T-75 flasks (catalog #430641) are recommended for subculturing this product.
  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.
Interval: Maintain cultures at a cell concentration between 2 X 104 and 2 X 105 cell/cm2.
Subcultivation Ratio: A subcultivation ratio of 1:2 to 1:6 is recommended
Medium Renewal: 2 to 3 times per week
Culture Conditions
Atmosphere: air, 95%; carbon dioxide (CO2), 5%
Temperature: 37°C
Cells per Vial Approximately 2 to 3 x 106 cells
Volume 1.0 mL
STR Profile
Amelogenin: X
CSF1PO: 11,12
D13S317: 11,14
D16S539: 9
D5S818: 12
D7S820: 9
THO1: 8
TPOX: 8,10
vWA: 16,17
Sterility Tests Bacteria and yeast: No growth
Mycoplasma: No growth
Viral Testing Hepatitis B: None detected
Cytomegalovirus: None detected
Human immunodeficiency virus: None detected
Epstein-Barr virus: None detected
Human papillomavirus: None detected
Functional Tests Resistance to BRAF inhibitor drugs.
Year of Origin 2016
References

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

Notice: Necessary PermitsPermits

These permits may be required for shipping this product:

  • Customers located in the state of Hawaii will need to contact the Hawaii Department of Agriculture to determine if an Import Permit is required. A copy of the permit or documentation that a permit is not required must be sent to ATCC in advance of shipment.
Basic Documentation
Other Documentation
FAQ's
  1. Gene editing technology for NRAS mutant-A375 Isogenic Cell Line ATCC CRL-1619IG-2
    The NRAS mutant - A375 Isogenic Cell Line (ATCC
    Date Updated: 2/1/2017
  2. Culture Conditions for ATCC CRL-1619IG-2 and A-375 ATCC CRL-1619
    No. The cell culture method and media for CRL-1619IG-2 are identical to its parental cell line, A-375 (ATCC® CRL-1619 ™).
    Date Updated: 2/1/2017
  3. Maintaining drug resistance in CRL-1619IG-2
    No. A375 Isogenic Cell Line (ATCC® CRL-1619IG-2 cell does not require supplementation of anti-B-Raf drugs in the culture media. This cell line is  heterozygous for the NRAS Q61K mutation...
    Date Updated: 2/1/2017
  4. Validated NRAS mutation in ATCC CRL-1619IG-2


    Date Updated: 2/1/2017

  5. Advantages of CRL-1619IG-2 as a melanoma model


    Date Updated: 2/1/2017

  6. Genetically engineered isogenic cell line
    An isogenic cell line is created by homologous genetic modification or genome editing of cells. It contains the same genetic composition as its parental line, differing only in the specific endog...
    Date Updated: 1/18/2016
Restrictions

For commercial accounts, this cell line is only distributed under the terms of a fully signed and executed ATCC® Material Transfer Agreement and Addendum. If the commercial account is screening per completed Addendum, the recipient will be required to pay a Screening Fee (ATCC® ACS-2103™) of $4000.00. Screening Use is defined as use of Biological Material in small molecule and biologic drug discovery, including initial target identification and validation, assay development, high throughput screening, hit identification, lead optimization, and selection of candidates for clinical development. If the commercial account is not screening per the completed Addendum, the recipient will not be required to pay a Screening Fee.

This material’s use is governed by the CRISPR Limited Use Label License. For information on obtaining a license to use this product for purposes other than those permitted in the Label License, please contact The Broad Institute at partnering@broadinstitute.org.  

References

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