In Vitro Expression of Human OTC in HeLa Cells

Example 14

To measure in vitro expression of human OTC in HeLa cells, those cells were seeded on 12-well plates (BD Biosciences, San Jose, USA) one day prior to transfection. mRNA formulations comprising human OTC or a GFP control was transfected using 800 ng mRNA and 2 μL Lipofectamin 2000 in 60 μL OPTI-MEM per well and incubated.

After 24 hours, the cells in each well were lysed using a consistent amount of lysis buffer. Appropriate controls were used, including citrate synthesase, a mitochondrial marker. Protein concentrations of each were determined using a BCA assay according to manufacturer's instructions. To analyze OTC expression, equal loads of each lysate (24 μg) were prepared in a loading buffer and subjected to standard Western blot analysis. For detection of OTC, a commercial anti-OTC antibody was used according to the manufacturer's instructions. The mRNA expressed OTC was compared to loaded recombinant human OTC protein (10, 5, and 2.5 ng).

FIG. 1A shows the expression level of human OTC (construct ahOTC; SEQ ID NO: 61) and the citrate synthesase mitochondrial protein control. FIG. 1B shows that the expressed human OTC (construct ahOTC; SEQ ID NO: 61) co-localizes with the mitotracker mitochondrial marker, and is therefore present in the mitochondria of cells.