The efficiency of three shRNAs in silencing the galactose-1-phosphate uridyl transferase gene

• Main Author: Nokoane, Mmateisi Patricia
• Other Authors: Pillay, M.
• Format: Others
• Language: en
• Published: 2015
• Subjects: GALT gene; RNA products; Enzyme activity; Escherichia coli; shRNA; Galactose-1-phosphate uridyl transferase gene; 660.6; Galactosemia; Metabolism > Disorders
• Online Access: http://hdl.handle.net/10352/230

M. Tech. (Biotechnology, Department of Biosciences, Faculty of Computer and Applied Sciences) Vaal University of Technology === This study seeks to design and test specific short hairpin RNA (pshRNA) for their efficiency in knocking down the GALT gene RNA products thereby limiting the resultant enzyme activity. The following objectives were followed in designing the current study: 1. Designing a shorthairpin RNA (pshRNA) to target different regions of the coding sequence of the target GALT gene. 2. Propagating the pshRNAs in Escherichia coli (E.coli) and subsequently isolation of the respective plasmids for transfection. 3. Transfection of HeLa cells to test the efficiency of relevant pshRNAs in knocking down the GALT gene expression. 4. Transfection was followed by extraction of total mRNA, purification and quantification of total mRNA. 5. The GALT gene expression was qualitatively quantified against a house-keeping gene, glyceraldehyde phosphate dehydrogenase (GAPDH) to evaluate efficiency of knockdown using real time PCR. The three newly designed pshRNA (pshRNA2, pshRNA3 and pshRNA4) targeting the GALT gene expression showed a knockdown efficiency of 171 %, 48 % and 200 %, respectively. The results of this study will be useful for future evaluation of the possible long term glycosylation patterns under proper UDP glucose/UDP galactose levels compared with variable defective GALT gene levels.