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 journalArticle

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

Fingerprint

small interfering RNA
RNA interference
green fluorescent protein
embryo (animal)
chickens
plasmids
fluorescence
gene silencing
reverse transcriptase polymerase chain reaction
cultured cells
microscopy
embryogenesis
protein synthesis
cells
fluorescent proteins

All Science Journal Classification (ASJC) codes

  • Animal Science and Zoology

Cite this

Application of RNA interference to chicken embryos using small interfering RNA. / Sato, Fuminori; Nakagawa, Tetsuto; Ito, Makoto; Kitagawa, Yasuo; Hattori, Masa Aki.

In: Journal of Experimental Zoology Part A: Comparative Experimental Biology, Vol. 301, No. 10, 01.10.2004, p. 820-827.

Research output: Contribution to journalArticle

Sato, Fuminori ; Nakagawa, Tetsuto ; Ito, Makoto ; Kitagawa, Yasuo ; Hattori, Masa Aki. / Application of RNA interference to chicken embryos using small interfering RNA. In: Journal of Experimental Zoology Part A: Comparative Experimental Biology. 2004 ; Vol. 301, No. 10. pp. 820-827.
@article{72b150ebe5814607a32b656cefb5ce27,
title = "Application of RNA interference to chicken embryos using small interfering RNA",
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.",
author = "Fuminori Sato and Tetsuto Nakagawa and Makoto Ito and Yasuo Kitagawa and Hattori, {Masa Aki}",
year = "2004",
month = "10",
day = "1",
doi = "10.1002/jez.a.99",
language = "English",
volume = "301",
pages = "820--827",
journal = "Journal of Experimental Zoology Part A: Comparative Experimental Biology",
issn = "1548-8969",
number = "10",

}

TY - JOUR

T1 - Application of RNA interference to chicken embryos using small interfering RNA

AU - Sato, Fuminori

AU - Nakagawa, Tetsuto

AU - Ito, Makoto

AU - Kitagawa, Yasuo

AU - Hattori, Masa Aki

PY - 2004/10/1

Y1 - 2004/10/1

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=7244254505&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=7244254505&partnerID=8YFLogxK

U2 - 10.1002/jez.a.99

DO - 10.1002/jez.a.99

M3 - Article

C2 - 15449340

AN - SCOPUS:7244254505

VL - 301

SP - 820

EP - 827

JO - Journal of Experimental Zoology Part A: Comparative Experimental Biology

JF - Journal of Experimental Zoology Part A: Comparative Experimental Biology

SN - 1548-8969

IS - 10

ER -