Establishment and application of a human osteosarcoma U-2OS cell line that can stably express Cas9 protein.

Abstract

Osteosarcoma is the most common primary malignant bone tumor, and U-2OS is a common osteosarcoma cell model. The study obtained a human osteosarcoma U-2OS tool cell line which could stably express Cas9 protein, and we reported its production method and application. Firstly, we introduced a Cas9 protein expression gene and an antibiotic screening marker gene through CRISPR/Cas9 system to construct a human osteosarcoma U-2OS tool cell line which could stably express Cas9 protein. Secondly, as the cell line could stably express Cas9 protein, it was only transfected alone a small sgRNA fragment for related gene editing, we then transfected, respectively, a small ETV4 and MALAT1 sgRNA fragment to U-2OS tool cell line for gene editing. Lastly, the Q-PCR results showed that the transcription levels of ETV4 and MALAT1 were significantly decreased, and western blotting result showed that the translation level of ETV4 was significantly decreased, these results indicated that the constructed U-2OS tool cell line could effectively edit protein-coding gene (ETV4) and long non-coding RNA gene (MALAT1). The results of this study also indicated that the constructed U-2OS tool cell line could greatly improve the efficiency of gene editing. Therefore, the genetic engineering cell line provided by the study is of great significance for studying the pathogenesis and regulatory network of osteosarcoma, and for preventing and treating bone tumor as soon as possible.