YUMMER1.7 was derived from YUMM1.7, which was generated from a cutaneous mouse melanoma containing the alleles BrafV600E, Pten−/−, Cdkn2a−/− (Meeth et al., 2016 (link)). Irradiation of YUMM1.7 included three rounds of 1500J/m2 UVB (3W for 500s) when cells were 50–70% confluent. Cells were given time to recover and proliferate before being re-plated and proceeding to the next UV treatment. After the final UV treatment, a single cell was clonally expanded. YUMM1.7 and YUMMER1.7 DNA content were assessed using a Propidium Iodide Flow Cytometry Kit according to manufacturer instructions (Abcam, Cambridge, UK). YUMMER1.7-GFP and YUMM1.7-GFP were generated using a P-YUK-GFP plasmid with PiggyBac Transposase Expression Vector, a gift from Tian Xu, Department of Genetics, Yale University. Transfection was done with Lipofectamine 2000 (Invitrogen, Carlsbad, California) and cells were selected using Blastomycin resistance. All cell lines were maintained in DMEM/F12 media containing 10% FBS and with 1% nonessential amino acids and 1% penicillin-streptomycin.
Protocol detail
Generating UV-Induced Melanoma Cell Lines
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Alleles
Amino acids
Blastomycin
Cdkn2a
Cell lines
Cells
Cutaneous melanoma
Flow cytometry
Irradiation
Lipofectamine 2000
Mouse
Penicillin
Plasmid
Propidium iodide
Pten
Streptomycin
Transfection
Transposase
Vector
Corresponding Organization : Yale University
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Variable analysis
independent variables
- Irradiation of YUMM1.7 with three rounds of 1500J/m^2 UVB (3W for 500s)
- YUMMER1.7 and YUMM1.7 DNA content assessed using Propidium Iodide Flow Cytometry Kit
- Generation of YUMMER1.7-GFP and YUMM1.7-GFP using P-YUK-GFP plasmid with PiggyBac Transposase Expression Vector
- Cells were given time to recover and proliferate before being re-plated and proceeding to the next UV treatment
- All cell lines were maintained in DMEM/F12 media containing 10% FBS and with 1% nonessential amino acids and 1% penicillin-streptomycin
- No positive or negative controls were explicitly mentioned in the provided information.
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