The role of HNF1B and PAX2 in normal kidney development : an experimental functional knocking down model using human embryonic kidney 293 (HEK293) cell lines

Abstract

PAX2 and HNF1B are involved in kidney development as mutations to these genes have been linked to the incidence of congenital anomalies of the kidney and urinary tract (CAKUT) (Zammit, 2018). This project aimed to investigate the roles PAX2 and HNF1B play in both the development of the kidney and nephrogenesis as well as the rise of CAKUT related diseases. The effects of PAX2 and HNF1B knock down on a set of genes present in HEK293 cells were observed. This simulated mutations occurring during early stage kidney and nephron development and allowed analysis of the CAKUT-related genotype-phenotype relationship for these genes. Cultured HEK293 cells were initially cultured in monolayer then transfected using HNF1B and PAX2 knocking down vectors transformed by DH5α E.coli. Next, RNA was extracted, converted to cDNA and run using qPCR. The fold expression change for each gene at each time point was then calculated Furthermore, gel electrophoresis was also conducted to ensure knock down of both genes. The fold expression change values obtained indicated changes in gene expression of all downstream genes. These values varied depending on which gene was initially knocked down by transfection and the time point post transfection. This shed light on the influence of PAX2 and HNF1B mutations on their downstream genes whilst additionally allowing CAKUT-related genotype-phenotype associations to be made.