hpgk1 luc reporter  (Addgene inc)

Journal: Molecular therapy. Methods & clinical development

Article Title: Design and validation of cell-based potency assays for frataxin supplementation treatments.

doi: 10.1016/j.omtm.2024.101347

Figure Lengend Snippet: Figure 3. Stable expression of exogenous human FXN in MUT MEFs (A) A schematic of the lentivirus vector pLenti hygro FXN encoding a human miniFXN gene. The miniFXN gene used in experiments contains fragments of the endogenous FXN 50UTR, promoter and intron 1, and all five exons of the gene. (B) Representative western blot demonstrating expression of exogenous FXN. The miniFXN encodes a C-terminal HA tag that results in a slightly greater molecular weight, as detected by western blot. (C) Quantitation of endogenous Fxn (*) and exogenous FXN by western blot. Gapdh was used as a normalization control. MUT+FXN designates cells expressing exogenous, human FXN. The experiment was performed three independent times. Results are plotted as mean values with standard deviations; (**p < 0.001, ***p < 0.0001).

Article Snippet: Cloning and packaging of pLenti hygro FXN-L A miniFXN construct expressing human FXN under the control of endogenous regulatory elements was cloned into the pLenti HRELuc pGK Hygro (Addgene plasmid #118706).

Techniques: Expressing, Plasmid Preparation, Western Blot, Molecular Weight, Quantitation Assay, Control

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Design and validation of cell-based potency assays for frataxin supplementation treatments

doi: 10.1016/j.omtm.2024.101347

Figure Lengend Snippet: Stable expression of exogenous human FXN in MUT MEFs (A) A schematic of the lentivirus vector pLenti hygro FXN encoding a human miniFXN gene. The miniFXN gene used in experiments contains fragments of the endogenous FXN 5′UTR, promoter and intron 1, and all five exons of the gene. (B) Representative western blot demonstrating expression of exogenous FXN. The miniFXN encodes a C-terminal HA tag that results in a slightly greater molecular weight, as detected by western blot. (C) Quantitation of endogenous Fxn (∗) and exogenous FXN by western blot. Gapdh was used as a normalization control. MUT+FXN designates cells expressing exogenous, human FXN. The experiment was performed three independent times. Results are plotted as mean values with standard deviations; (∗∗ p < 0.001, ∗∗∗ p < 0.0001).

Article Snippet: A fragment of 2,782 base pairs (bp) containing the entire FXN gene was excised from pAAV FXN-L using Mlu I, Pml I, and Aat I restriction enzyme digestion and cloned into pLenti HRE-Luc pGK Hygro digested with Mlu I and Pme I. Lentivirus packaging was performed in HEK293 cells using pMD2.G (Addgene #12259) and psPAX2 (Addgene #12260), together with the pLenti hygro FXN-L.

Techniques: Expressing, Plasmid Preparation, Western Blot, Molecular Weight, Quantitation Assay, Control