Application of RNA interference to chicken embryos using small interfering RNA

Fuminori Sato, Tetsuto Nakagawa, Makoto Ito, Yasuo Kitagawa, Masa Aki Hattori

Research output: Contribution to journalArticlepeer-review

17 Citations (Scopus)

Abstract

Gene silencing using small interfering RNA (siRNA) is not established in avian species. The present study was performed to evaluate RNA interference (RNAi) in the chicken embryo by using a dual fluorescence reporter assay, a plasmid encoding green fluorescent protein (GFP) and a plasmid encoding red fluorescent protein (RFP). The siRNA targeting the GFP mRNA sequence (GFP-siRNA) with both plasmids was introduced into cultured cells and whole embryos by lipofection and microelectroperation, respectively. GFP- and RFP-expressed cells and embryos were observed under fluorescent microscopy and analyzed by flow cytometer, and their mRNAs were analyzed by reverse transcription-PCR (RT-PCR). The strong fluorescence was observed by introducing both plasmids into cells. The intensity of the green fluorescence generated by GFP was greatly suppressed by introducing GFP-siRNA. RT-PCR analysis showed that introducing GFP-siRNA also decreased GFP mRNA levels. In contrast to GFP, the intensity of the red fluorescence generated by RFP and the RFP mRNA levels remained unchanged. In whole embryos, also, introducing GFP-siRNA specifically suppressed GFP expression, and the suppression was maintained for at least 72 h. Consequently, it was concluded that the gene silencing using siRNA is applicable to analyzing the function of genes of interest during avian embryogenesis.

Original languageEnglish
Pages (from-to)820-827
Number of pages8
JournalJournal of Experimental Zoology Part A: Comparative Experimental Biology
Volume301
Issue number10
DOIs
Publication statusPublished - Oct 1 2004

All Science Journal Classification (ASJC) codes

  • Animal Science and Zoology

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