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RNAi‐mediated inhibition of COL1A1 and COL3A1 in human skin fibroblasts
Author(s) -
Wang Qiong,
Peng Zhenhui,
Xiao Shengxiang,
Geng Songmei,
Yuan Jingyi,
Li Zhengxiao
Publication year - 2007
Publication title -
experimental dermatology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.108
H-Index - 96
eISSN - 1600-0625
pISSN - 0906-6705
DOI - 10.1111/j.1600-0625.2007.00574.x
Subject(s) - rna interference , gene knockdown , small interfering rna , transfection , microbiology and biotechnology , extracellular matrix , biology , cell culture , connective tissue , chemistry , rna , gene , genetics
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).