Figure 1.

Codon optimization of the hOTC cDNA increases protein production without affecting sub-cellular localization

(A) Scheme of the rAAV-hOTC expression cassette. ITR, AAV2 inverted terminal repeat; intron, modified hemoglobin (HBB2) beta intron; LSP, liver-specific promoter (ApoE/hAAT, hybrid promoter containing ApoE enhancer and hAAT promoter); hOTC, WT or CO hOTC open reading frame (ORF); pA, hemoglobin beta (HBB) polyadenylation signal; the wavy lines indicate AAV backbone sequences between the expression cassette and ITRs. (B) Analysis of HCC cell line Huh-7 transfected with first group of hOTC-CO constructs. Representative western blot analysis of OTC protein levels in Huh-7 cell lysates (15 μg of protein per lane) following co-transfection of the pSMD2-hOTC-WT, pSMD2-hOTC-CO01, pSMD2-hOTC-CO21, and pSMD2-hOTC-CO03 constructs and GPF plasmids. The control lane contains cell lysate from cells transfected with the GFP-expressing plasmid. (C) Densitometric quantification of hOTC proteins from (B). GFP was used as transfection control. Values are expressed as fold change with respect to the pSMD2-hOTC-WT plasmid. Data are shown as mean ± SEM, and statistical analyses were performed by one-way ANOVA with Turkey’s multiple comparison test (n = 2, ∗p < 0,05). (D) Sub-cellular localization studies of hOTC in Huh-7 human liver cells. Huh-7 cells were transfected with plasmids encoding the hOTC-CO21 variant. Mitochondria (red, MITO-TRACK) and hOTC (green) were detected with a confocal microscope. A magnified picture of the indicated area is shown below the low-resolution picture. The scale bar corresponds to 14 μm.