4.1. Stable HEK-293 Cell Line Generation

The ORF expression clone was obtained from GeneCopoeia (pEZ_M98), which encoded RDH12 ({"type":"entrez-nucleotide","attrs":{"text":"NM_152443","term_id":"1653962297","term_text":"NM_152443"}}NM_152443) with a C-terminal GFP tag. Three known patient mutations (c.677A > G, p.Y226C; c.325G > C, p.A109P; and c.38C > A, p.S13*) were introduced into plasmids using the Quickchange II Site-Directed Mutagenesis kit (Agilent, Santa Clara, CA, USA). Primers were designed, according to kit instructions, with approximately 10 bases of correct sequence either side of the mutation (Supplementary Materials Table S1). Colonies with the correct mutation were verified by Sanger sequencing.

HEK-293 cells were cultured in DMEM high glucose, 10% FBS, and penicillin/streptomycin (Thermo Fisher Scientific, Waltham, MA, USA), and medium was changed every 3–4 days. Cells were passaged at 80–90% confluency using TrypLE Express (Thermo Fisher Scientific).

Cells were plated in six-well plates at a density of 600,000 cells per well and, 24 h later, they were transfected using Lipofectamine 2000 Reagent (Thermo Fisher Scientific). Twenty-four hours after transfection, cells were passaged into 96-well plates in selection media (DMEM high glucose, 10% FBS, 1 mg/mL G418) using serial dilutions to achieve low cell density. After 10 days, all non-transfected cells died off and, after a further 2 weeks, transfected cells formed distinct colonies. Individual colonies were picked into 24-well plates and expanded. This was defined as passage 0. For continued culture, G418 concentration was reduced to 0.5 mg/mL. All further experiments were carried out on cells from passage 5 onwards.