RNAi-mediated inhibition of COL1A1 and COL3A1 in human skin fibroblasts.
Types I and III collagens are the major collagens comprising skin connective tissue. Defects in these collagens lead to diseases of dermal connective tissue and fibre hyperplasia. RNA interference (RNAi) provides a powerful tool to inhibit specific gene expression. In this study, we generated small interfering RNAs (siRNA) expression cassettes (SECs) by polymerase chain reaction (PCR) as a method to quickly screen the efficacy of siRNAs. We then cloned the most efficient SECs into vectors, using a rapid and novel method intrinsic to the design of the SEC, and transfected human skin fibroblasts (HSF) to generate stable lines. We show that the transfection of SECs into HSFs resulted in specific and effective repression of COL1A1 and COL3A1 expression (5.00% and 6.48% of control levels) provided a rapid method for testing candidate siRNA sequences. We report the use of vector-based RNAi to establish stable HSF cell lines with persistent knockdown over at least 30 days (25.21% and 22.12% of control levels). These stably modified HSF cell lines may be used for the study of other types of collagen or proteins of the extracellular matrix (ECM).
Wang
Qiong
Q
Department of Dermatology, Second Hospital of Xi'an Jiaotong University, Shaanxi, China.
Peng
Zhenhui
Z
Xiao
Shengxiang
S
Geng
Songmei
S
Yuan
Jingyi
J
Li
Zhengxiao
Z
D015153:Blotting, Western; D002460:Cell Line; D049109:Cell Proliferation; D024042:Collagen Type I / Q000235:genetics* / Q000378:metabolism; D000090602:Collagen Type I, alpha 1 Chain; D024061:Collagen Type III / Q000235:genetics* / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism* / Q000473:pathology / Q000502:physiology; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006801:Humans; D008055:Lipids / Q000235:genetics; D009841:Oligonucleotides / Q000235:genetics; D016133:Polymerase Chain Reaction; D034622:RNA Interference; D014162:Transfection / Q000379:methods
C439971:COL3A1 protein, human; D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D024061:Collagen Type III; D008055:Lipids; C086724:Lipofectamine; D009841:Oligonucleotides