Plasmid Construction and Expression Studies
Names and inserts of plasmids used in this study are listed in Table 1. Plasmid p4.23hE (Heibel et al., 2011), renamed minP-E in this study (Table 1), was the template for introduction of theNAGS :c.-3065A>C andNAGS :c-3098C>T sequence changes using mutagenic primers 5’-tcc ctc cga cct ggg act ccg gga c-3’ and 5’-gtc ccg gag tcc cag gtc gga ggg a-3’ for the c.-3065A>T variant and 5’-cat tga ccc tgg gac cac tgt gtc ccc-3’ and 5’-ggg gac aca gtg gtc cca ggg tca atg-3’ for the c.-3098C>T variant. Site directed mutagenesis was performed using QuickChange Site-Directed Mutagenesis kit (Agilent Technologies) according to manufacturer’s instructions. Resulting plasmids were called c.-3065A>C and c.-3098C>T (Table 1), and the presence of the correct sequence changes were verified with DNA sequencing.
Plasmid Prom-Ex1-Int1-Luc (Sonaimuthu et al., 2021) was a template for introduction of the NAGS :c.426+326G>A and c.427-218A>C sequence changes. Mutagenic primers 5’-ccg acc ttg gat tcc ggg aca tct ttg ggg g-3’ and 5’-ccc cca aag atg tcc cgg aat cca agg tcg g-3’ were used to generate theNAGS :c.426+326G>A sequence change while primers 5’-tcc ctc cga cct ggg act ccg gga c-3’ and 5’-gtc ccg gag tcc cag gtc gga ggg a-3’ were used for the c.427-218A>C sequence change. The QuickChange Site-Directed Mutagenesis kit (Agilent Technologies) was used to introduce the mutations according to manufacturer’s instructions; resulting plasmids were called c.426+326G>A and c.427-218A>C (Table 1) and their correct sequence was verified with DNA sequencing.
Culturing of the HepG2 cells (ATCC) and the HuH-7 cells (Charles Rice laboratory, Rockefeller University), their transfections and reporter assays were carried out as described previously (Sonaimuthu et al., 2021; Williams et al., 2018).