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PMID:17576241

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Citation

Wang, Q, Peng, Z, Xiao, S, Geng, S, Yuan, J and Li, Z (2007) RNAi-mediated inhibition of COL1A1 and COL3A1 in human skin fibroblasts. Exp. Dermatol. 16:611-7

Abstract

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).

Links

PubMed Online version:10.1111/j.1600-0625.2007.00574.x

Keywords

Blotting, Western; Cell Line; Cell Proliferation; Collagen Type I/genetics; Collagen Type I/metabolism; Collagen Type III/genetics; Collagen Type III/metabolism; Fibroblasts/metabolism; Fibroblasts/pathology; Fibroblasts/physiology; Gene Expression Regulation; Genetic Vectors; Humans; Lipids/genetics; Oligonucleotides/genetics; Polymerase Chain Reaction; RNA Interference; Transfection/methods

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

HUMAN:CO1A1

part_of

GO:0005584: collagen type I trimer

ECO:0000315: mutant phenotype evidence used in manual assertion

C

Seeded From UniProt

complete

HUMAN:CO3A1

part_of

GO:0005586: collagen type III trimer

ECO:0000315: mutant phenotype evidence used in manual assertion

C

Seeded From UniProt

complete

See also

References

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