Establishment of MageC2-knockout cells through CRISPR/CAS9 technology

FATIMA LEDELFA; MICAELA ESCALADA ; FRANCO PASCUCCI; MONTE MARTIN

Mar del Plata

Congreso; XIX Reunión Científica anual de la Sociedad Científica Argentina De Investigación Clínica (SAIC); 2019

MageC2 is a member of the melanoma antigen gene (MAGE) family, specifically expressed in awide variety of human cancers and associated with a non-favourable clinical course. It has beenreported that MageC2 oncoprotein downregulates p53 and activates STAT3 transcription factors.Recently, we identified the Ras oncogene as responsible for MageC2 stability increase andphosphorylation through the MEK/ERK pathway. Then, our hypothesis is that Ras oncogene couldregulate p53 and STAT3 by enhancing MageC2 protein levels.In order to analyze the functional consequences of endogenous MageC2 expression, weestablished a MageC2 knockout (KO) cell line through the CRISPR/Cas9 system in humanmelanoma A375 cells. All the obtained clones were probed for MageC2 protein expression byWestern blot. Three clones were selected and sequenced for indel detection (KO1, gRNA1; KO2,gRNA1 and KO3, gRNA2). To validate the A375 MageC2 KO (A375-C2KO) clone behaviour, wequantified the mRNA levels of genes regulated by p53 or STAT3. Analysis of RT-qPCR data carriedout in three pairs of biological replicates in A375-C2KO cells indicated enhanced transcription ofp53 targets (p21waf1 and bax, p