Hussein Nasserdine
Pronouns:
Research Mentor(s): Xin Tong
Research Mentor School/College/Department: Molecular & Integrative Physiology / Medicine
Program: UROPF
Session: Session 7 (4:40pm – 5:30pm)
Authors: Xin Tong, Hussein Nasserdine, Joel Groves
Abstract
Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease, afflicting over 30% of adults in the United States. So far, no FDA-approved treatment is available. To study the impact of various genetic factors on the development and progression of NAFLD towards non-alcoholic steatohepatitis and liver fibrosis, researchers routinely over-express target genes in the liver via adenovirus-mediated delivery. To ensure the specificity of the effects of target genes, we need a control adenovirus that expresses a gene that is unlikely to affect the liver function and the pathogenesis of NAFLD. To this end, we generated an adenoviral stock that over-expresses a FLAG-tagged bacterial enzyme, ß-galactosidase, which is encoded by the LacZ gene. First, the Flag-tagged LacZ coding sequence was amplified by PCR and then subcloned into the pDONR/Zeo empty vector via Gateway BP homologous recombination. The insertion of Flag-LacZ was verified by PCR screening, restriction enzyme digestion, and direct sequencing. Lastly, the Ad-Flag-LacZ construct was generated through Gateway LR recombination and selected by restriction enzyme digestion. After transfection of 293AD packaging cells, Ad-Flag-LacZ virus was produced, amplified, and eventually concentrated by ultra-centrifuge in CsCl gradient. The expression of FLAG-tagged LacZ was confirmed by staining, enzymatic assay, and immunoblotting. Taken together, our project has successfully generated and validated Ad-Flag-LacZ as a control virus for studying NAFLD.
