wt construct  (Addgene inc)

Journal: JCI Insight

Article Title: Splicing variants in MYRF cause partial loss of function in the retinal pigment epithelium leading to nanophthalmos

doi: 10.1172/jci.insight.194681

Figure Lengend Snippet: ( A ) Diagram of amino acid changes in WT-MYRF, dG-MYRF, and cleavage deficient variant V679A-MYRF. ProRich, proline rich; DBD, DNA binding domain; ICA, intramolecular chaperone auto-processing; TM, transmembrane; C2, C-terminal. ( B ) Localization of FLAG-tagged MYRF constructs in ARPE-19 cells show normal nuclear localization of dG-MYRF ( n = 3). Scale bar: 50 μm. ( C ) Western blot of transfected ARPE-19 cells shows no change in cleavage of N-terminal fragment ( n = 3). ( D ) qPCR analysis of RNA from ARPE-19 cells transduced with dG-MYRF, compared with WT-MYRF show decreased levels of transcripts for total Myr f and endogenous Tmem9 8 mRNA ( n = 3) by Student’s t test. * P < 0.05, ** P < 0.01.

Article Snippet: Human FLAG-tagged WT-MYRF DNA constructs were generated (Twist Bioscience, South San Francisco, CA) in a pLentiLox-IRES-GFP backbone vector ( ).

Techniques: Variant Assay, Binding Assay, Construct, Western Blot, Transfection, Transduction

Journal: JCI Insight

Article Title: Splicing variants in MYRF cause partial loss of function in the retinal pigment epithelium leading to nanophthalmos

doi: 10.1172/jci.insight.194681

Figure Lengend Snippet: ( A – C ) Localization of FLAG-tagged MYRF constructs in mature human primary RPE show normal nuclear localization of dG-MYRF. Scale bar: 5 μm. ( D ) Western blot of transduced human primary RPE cells shows no change in cleavage of N-terminal fragment. ( E ) Functional assessment of barrier function via TEER (normalized to pretransduction TEER value for each replicate) shows no significant difference in TEER 1 week after transduction with Empty Vector, WT-MYRF, or dG-MYRF by 1-way ANOVA (all experiments are 3 reps from n = 2 donors).

Article Snippet: Human FLAG-tagged WT-MYRF DNA constructs were generated (Twist Bioscience, South San Francisco, CA) in a pLentiLox-IRES-GFP backbone vector ( ).

Techniques: Construct, Western Blot, Functional Assay, Transduction, Plasmid Preparation

Journal: JCI Insight

Article Title: Splicing variants in MYRF cause partial loss of function in the retinal pigment epithelium leading to nanophthalmos

doi: 10.1172/jci.insight.194681

Figure Lengend Snippet: WT MYRF retains transcription factor function resulting in no phenotypic effects. A C-terminal frameshift splice variant results in a 31 amino acid frameshift leading to decreased C-terminus stability and transcriptional activation, leading to reduced transcription factor function and isolated ocular disease. Intronic splicing variants lead to a frameshift that results in early truncation or nonsense mediated RNA decay. These splice sites are not always used, leading to modest decrease in MYRF transcription factor function and isolated ocular disease. In contrast, complete loss of function alleles either lead to early truncation/nonsense-mediated decay or alter critical functional domains (i.e., DNA binding, cleavage) and lead to severe, syndromic symptoms.

Article Snippet: Human FLAG-tagged WT-MYRF DNA constructs were generated (Twist Bioscience, South San Francisco, CA) in a pLentiLox-IRES-GFP backbone vector ( ).

Techniques: Variant Assay, Activation Assay, Isolation, Functional Assay, Binding Assay