Non-Invasive Intravital Imaging of siRNA-Mediated Mutant Keratin Gene Repression in Skin
| Autor: | Robyn P. Hickerson, Manuel A. Flores, Tycho Speaker, Christopher H. Contag, Emilio Gonzalez-Gonzalez, Maria Fernanda Lara, Roger L. Kaspar |
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| Rok vydání: | 2015 |
| Předmět: |
0301 basic medicine
Cancer Research Small interfering RNA Injections Intradermal Green Fluorescent Proteins Mutant Gene Expression Biology medicine.disease_cause Cell Line Mice Protein Aggregates 03 medical and health sciences Genes Reporter Complementary DNA Keratin medicine Animals Humans Pachyonychia congenita Radiology Nuclear Medicine and imaging RNA Small Interfering Skin chemistry.chemical_classification Regulation of gene expression Mutation Wild type medicine.disease Molecular biology Molecular Imaging Repressor Proteins 030104 developmental biology Oncology chemistry Keratins Mutant Proteins Plasmids |
| Zdroj: | Molecular Imaging and Biology. 18:34-42 |
| ISSN: | 1860-2002 1536-1632 |
| Popis: | Small interfering RNAs (siRNAs) specifically and potently inhibit target gene expression. Pachyonychia congenita (PC) is a skin disorder caused by mutations in genes encoding keratin (K) 6a/b, K16, and K17, resulting in faulty intermediate filaments. A siRNA targeting a single nucleotide, PC-relevant mutation inhibits K6a expression and has been evaluated in the clinic with encouraging results. To better understand the pathophysiology of PC, and develop a model system to study siRNA delivery and visualize efficacy in skin, wild type (WT) and mutant K6a complementary DNAs (cDNAs) were fused to either enhanced green fluorescent protein or tandem tomato fluorescent protein cDNA to allow covisualization of mutant and WT K6a expression in mouse footpad skin using a dual fluorescence in vivo confocal imaging system equipped with 488 and 532 nm lasers. Expression of mutant K6a/reporter resulted in visualization of keratin aggregates, while expression of WT K6a/reporter led to incorporation into filaments. Addition of mutant K6a-specific siRNA resulted in inhibition of mutant, but not WT, K6a/reporter expression. Intravital imaging offers subcellular resolution for tracking functional activity of siRNA in real time and enables detailed analyses of therapeutic effects in individual mice to facilitate development of nucleic acid-based therapeutics for skin disorders. |
| Databáze: | OpenAIRE |
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