Study the optimal condition to knockout metallothionein by CRISPR-Cas9 in neuroblastoma

Májková, Klára

Cisplatin-based treatment strategies are commonly employed for the treatment of Neuroblastoma (Nbl), an embryonal tumour that most commonly affects infants and children under the age of five. However, the efficacy of these strategies has been found to be lower than 50 % due to the frequent chemoresistance developed by the tumour cells. Among others, this resistance has been linked to the increased expression of metallothionein (MTs) by the Nbl cells. Human MT-3, a protein primarily expressed in central nervous system cells, plays a vital role in metal detoxification and the maintenance of homeostasis during oxidative stress. Therefore, knockout of MT-3 using CRISPR/Cas9 could make the cells sensitive to cytotoxic drugs. This study is aimed to optimize the conditions for successful knockout of MT-3 from Nbl cells. The MT3-CRISPR/Cas9 plasmid was transfected into Nbl using Lipofectamine 2000 in three different plasmid concentrations (250 ng/μL, 500 ng/μL, and 1000 ng/μL). The optimal conditions were achieved using 0.75 μl of Lipofectamine, 1000 ng/μl of plasmid, and 0.5 ng/μl of puromycin.

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