In-vitro SMA model; SMN1 gene knockdown in SH-SY5Y cells

Abstract

Spinal muscular atrophy is an autosomal­recessive disorder characterized by degeneration of motor neurons in the spinal cord and caused by mutations in the survival motor neuron 1 gene, SMN1. The phenotype is extremely variable, and patients have been classified in type I­ II and III. SMA based on age at onset and clinical course. All three types of SMA are caused by mutations in the survival motor neuron gene (SMN1). Therefore, in this study we aimed to knockdown of SMN1 gene in Human Neuroblastoma cells to generate SMA model for in­ vitro studies. For this aim, target sequences of SMN1 for two regions were analyzed using bioinformatic tools and these target sequences were cloned into shRNA­based vector system (pLKO.1). Fist, the vector was restricted with EcoRI and AgeI restriction enzymes. Then, the primers were annealed and ligated into EcoRI and Age I restricted pLKO.1 vector. Transformation of ligation reaction was performed into DH5α competant cells. The colonies were picked and plasmid isolation was performed using plasmid isolation kit (Mn). Recombinant colonies were screened using restriction procedure with EcoRI and NcoI enzymes. The recombinant colonies were verified with sequence analyses. In a result, we obtained two plasmid vectors namely pLKO.1shRNA1 (target region 1) and pLKO.1 shRNA2 (target region2). The vectors were transfected using Turbofect transfection reagent as manufacturers’instructions. Puromycin selection was carried out after 48 h of transfection. Then the pellet of cells were used for RNA isolation and cDNA was obtained from RNA templates. The mRNA level of SMN1 was determined with Real­Time PCR analyses. We obtained that, the mRNA level of SMN1 was decreased approximately 25 fold in SMN1 knockdown ShSy­5y cells when compared to control cells (SMN1 positive).