Journal: Molecular Therapy. Nucleic Acids
Article Title: RNAi-mediated Gene Silencing of Mutant Myotilin Improves Myopathy in LGMD1A Mice
Figure Lengend Snippet: miMYOT mediates silencing of human myotilin in vitro and in vivo . ( a ) Four different MYOT-targeted artificial miRNAs (mi1291, mi1321, mi1366, and mi1490) were generated and tested for their ability to stimulate human MYOT gene silencing in HEK293 cells. Data shown are representative of three independent experiments. The lead miMYOT construct, mi1321, stimulated an 81 and 62% knockdown of MYOT mRNA (top, real-time PCR) and protein (bottom, western blot), respectively. Gene silencing data are calculated relative to samples transfected with a control miRNA targeting eGFP (miGFP). 22 Individual samples were normalized to GAPDH. The mi1321 construct is referred to as miMYOT in all subsequent figures. ( b,c ), AAV6-delivery of the U6.miMYOT construct to TgT57I mouse gastrocnemius muscle reduced mutant MYOT expression in 3- and 9-month-old animals, compared to AAV6-treated miGFP or miLacZ 29 controls, respectively. Specifically, by 3 months, MYOT mRNA and protein was reduced 50 and 54% and at 9 months, these values were 79 and 63%, respectively. For both timepoints, N = 8 animals, with one leg receiving AAV.miMYOT and the other a control miRNA vector. QPCR data represents means ± SEM. Western blots show three representative samples. C, control-treated legs; T, miMYOT-treated legs.
Article Snippet: Mice hemizygous for human myotilin TgT57I transgene were purchased from Jackson Laboratory (Bar Harbor, ME) and maintained by breeding onto the C57BL/6 background.
Techniques: In Vitro, In Vivo, Generated, Construct, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Mutagenesis, Expressing, Plasmid Preparation