Non-Invasive Intravital Imaging of siRNA-Mediated Mutant Keratin Gene Repression in Skin.
Non-Invasive Intravital Imaging of siRNA-Mediated Mutant Keratin Gene Repression in Skin.
复制标题
siRNA 介导的皮肤突变角蛋白基因抑制的非侵入性活体成像。
DOI：
10.1007/s11307-015-0875-z
复制
发表时间：
2016
影响因子：
3.1
通讯作者：
Kaspar,RogerL
中科院分区：
文献类型：
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作者：
Hickerson,RobynP;Speaker,TychoJ;Lara,MariaFernanda;González-González,Emilio;Flores,ManuelA;Contag,ChristopherH;Kaspar,RogerL
目的小分子干扰RNA（siRNA）能特异性地抑制靶基因的表达。先天性甲肥厚（PC）是一种皮肤病，由编码角蛋白（K）6a/B、K16和K17的基因突变引起，导致中间丝缺陷。靶向单个核苷酸的siRNA，PC相关突变抑制K6 a表达，并且已经在临床上进行了评估，结果令人鼓舞。野生型（WT）和突变型K6 a互补DNA（cDNA）与增强型绿色荧光蛋白或串联番茄荧光蛋白cDNA融合，以允许使用配备有双荧光的体内共聚焦成像系统在小鼠足垫皮肤中共观察突变体和WT K6 a表达结果突变型K6 a/报告基因的表达导致角蛋白聚集体的可视化，而WT K6 a/报告基因的表达导致角蛋白掺入到细丝中。添加突变体K6 a特异性siRNA导致突变体的抑制，但不是WT，K6 a/reporter expression. ConclusionsIntravitiveimaging提供亚细胞分辨率为跟踪siRNA的功能活性在真实的时间，并使详细的分析在个别小鼠的治疗效果，以促进开发基于核酸的治疗皮肤疾病。
PurposeSmall 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.ProceduresTo 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 fluorescencein vivoconfocal imaging system equipped with 488 and 532 nm lasers.ResultsExpression 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.ConclusionsIntravital 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.