DSCR1 gene silencing by siRNA in lymphoblast cells from patient with down syndrome

Abstract

DSCR1 (Down Syndrome Critical Region 1) gene directly affect to the learning and memory process in human expecially in fetal Down Syndrome (DS) brain. DSCR1 belongs to a highly conserved calcineurin inhibitor family called calcipressin. DSCR1 can bind to and inhibit calcineurin, a protein important for learning and memory. RNAi technique which is a naturally occurring cellular mechanism that induces post transcriptional gene silencing .Small interfering RNA (siRNA) molecules are the key intermediaries in post transcriptional gene silencing which when exogenously administered can inhibit the expression of any given target gene. Since DSCR1 is overexpressed in DS fetal brain, it is possible that normalizing DSCR1 expression may restore normal brain function in DS individual. The goal of this study is to inhibit DSCR1 gene in lymphoblast cells by siRNA. In this research we studied DSCR1 gene expression level in both mRNA and protein by real time PCR and western blot consequently.The comparison result of mRNA level between untreated and treated with 3 concentrations of siRNAs ( 0.4 ,0.8 and 1 μg) in control and case samples indicated that siRNAs did not affect to mRNA level of DSCR1 gene in both of samples at 14th day (Pr=0.7878, 0.7099 and 0.4103) From above result it might be the long period of post-transfection. The DsRed2 gene was cloned into siRNA plasmid vector to indicate the transfection efficiency. RFP signal was shown that siRNAs were effectively within 8 days of post transfection .From this result , I decide to make an additional experiment by decrease time period and sample quantity, harvest lymphoblast cell lines 5 samples from normal and measure mRNA at 5th day of post-transfection at appropriate siRNA concentration (0.8 μg) for test DSCR1 suppression . After changing the time, I found siRNA cannot knockdown DSCR1 gene (t test, Pr=0.3431), Moreover we tested transfection efficiency in both of fibroblast and lymphoblast cells from same case sample. We demonstrate that the percentage of transfection efficiency and period of signal in both of cell types are not different .