Model Generation
Creators: name: Llinàs Arias, Pere
Publisher: name: figshare
Year: 2023
Subjects: Cancer genetics
Cancer cell biology

description: <strong>Cell culture growth and handling </strong> The human triple-negative breast cancer cell lines MDA-MB-231 and MDA-MB-436 were purchased at the American Type Culture Collection (ATCC). Cells were maintained at 37ºC in 5% CO2 between 20-80% confluency in RPMI 1640 w/glutamine (Biowest, L0500-500) complete media, which contained 10% fetal bovine serum (Biowest, S181BH-500), and 1% penicillin/streptomycin (Biowest, L0018-100). When cells reached 70-80% confluency, media was removed, and cells were gently washed with 1x PBS. After PBS removal, the appropriate amount of trypsin (ThermoFisher Scientific, R001100; 50uL in 96-well plates, 200 in p24-well plates, 500 in 6-well plates, 1mL in T25 flasks and 1.5mL in T75 flasks) was added to the wells/flasks. Cells were then incubated for 3-5 minutes at 37ºC. Cells were gathered by washing the plate with at least 4 volumes of the complete media. Cells were collected in a 15 mL tube and centrifuged for 5 minutes at 500 x g. After centrifugation, media containing trypsin was removed, and cell pellets were resuspended in complete media. A fraction of the dilution was used to maintain the culture (1/4 to 1/2 dilutions were typically performed). During cell expansion, either after thawing or after limit dilution to generate clones, all the cells were collected and seeded in a larger plate sequentially until they were expanded into T75 flasks. Short tandem repeat analysis was used by the Genetics Core at the University of Arizona to validate cell lines (Phoenix, USA). Using the MycoAlert Mycoplasma Detection Kit, all cell lines and models were periodically checked to ensure they were free of mycoplasma contamination (Lonza, lt07-418). <strong>Generation of IE8 deficient stable cell line models</strong> Our experimental approach consisted in generating stable cell lines deficient in the insulator element of our interest, the IE8 (chr11: 102,730,781-102,736,005) (Figure 1). To do that, we transiently transfected both MDA-MB-231 and MDA-MB-436 cell lines with either the empty PsPCas9pSpCas9(BB)-2A-GFP (PX458) vector or the PX458 containing a sgRNA designed using CHOPCHOP10 against IE8 (Fd: CACCGTCCACTGGGGTATTCGGA, Rv: AAACTCCGAATACCC-CAGTGGAC). PX458 was a gift from Feng Zhang lab (Addgene # 48138). Plasmid sgRNA incorporation into pspCas9 was performed as described by Ran F, et al11. After that, the plasmid sequence was verified by Sanger sequencing. Cells were transfected following the manufacturer’s instructions using Lipofectamine 3000 (ThermoFisher Scientific, L3000001). Optimem (ThermoFisher Scientific, 31985062), Lipofectamine 3000, P3000 reagent, and plasmid volumes were scaled considering the T25 flask surface. 48h after transfection, GFP-positive cells were sorted using a BD FACSAria Fusion sorter. GFP-positive cells transfected with the empty PX458 plasmid were collected and maintained as the wild-type population. Contrarily, GFP-positive cells transfected with sgRNA-containing plasmid were serially diluted in 96-well plates to isolate single clones. 48h after seeding, 96-well plates were examined to identify which wells contained a single cell. Cells were incubated in conditioned media (50% of complete media and 50% of complete media collected from parental TNBC cell lines filtered using 0.22um filters (Meck Millipore, SLGVV255F)). Cells located in those wells were expanded for 20-30 days. Once expanded, the CRISPR/Cas9-mediated disruption was interrogated by Sanger sequencing (Macrogen, Madrid). Briefly, DNA was purified using Quick-DNA Microprep Kit (Zymo Research, Cat D3020). DNA amplicon harboring the sgRNA target sequence was amplified by PCR (Fd: CCCTACTTCAGTCCCCTCT; Rv: TTCAAAACCTGGGCTCATCC), purified using NucleoSpin Gel and PCR clean-up kit (Macherey-Nagel) and sequenced (TTCAATGAAGTCACGGATGG). PCR primers used for amplicon generation and sequencing were designed using Primer3 webtool12. A single clone for each cell line that contained <em>indels</em> at the IE8 locus was selected for further experiments.