Properties of gene knockdown system by vector-based siRNA in zebrafish

Minori Shinya, Kayo Kobayashi, Aki Masuda, Mika Tokumoto, Yuichi Ozaki, Kenji Saito, Toshihiro Kawasaki, Yukiko Sado, Noriyoshi Sakai

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

RNA interference (RNAi) has emerged as a powerful tool to silence specific genes. Vector-based RNAi systems have been developed to downregulate targeted genes in a spatially and temporally regulated fashion both in vitro and in vivo. The zebrafish (Danio rerio) is a model animal that has been examined based on a wide variety of biological techniques, including embryonic manipulations, forward and reverse genetics, and molecular biology. However, a heritable and tissue-specific knockdown of gene expression has not yet been developed in zebrafish. We examined two types of vector, which produce small interfering RNA (siRNA), the direct effector in RNAi system; microRNA (miRNA) process mimicking vectors with a promoter for RNA polymerase II and short hairpin RNA (shRNA) expressing vector through a promoter for RNA polymerase III. Though gene-silencing phenotypes were not observed in the miRNA process mimicking vectors, the transgenic embryos of the second vector (Tg(zU6-shGFP)), shRNA expressing vector for enhanced green fluorescence protein, revealed knockdown of the targeted gene. Interestingly, only the embryos from Tg(zU6-shGFP) female but not from the male fish showed the downregulation. Comparison of the quantity of siRNA produced by each vector indicates that the vectors tested here induced siRNA, but at low levels barely sufficient to silence the targeted gene.

Original languageEnglish
Pages (from-to)755-765
Number of pages11
JournalDevelopment Growth and Differentiation
Volume55
Issue number9
DOIs
Publication statusPublished - 2013 Dec
Externally publishedYes

Fingerprint

Gene Knockdown Techniques
Zebrafish
Small Interfering RNA
RNA Interference
MicroRNAs
Down-Regulation
Embryonic Structures
Genes
RNA Polymerase III
Reverse Genetics
RNA Polymerase II
Gene Silencing
Molecular Biology
Fishes
Animal Models
Fluorescence
Phenotype
Gene Expression
Proteins

Keywords

  • RNAi
  • ShRNA
  • SiRNA
  • U6 snRNA promoter

ASJC Scopus subject areas

  • Developmental Biology
  • Cell Biology

Cite this

Shinya, M., Kobayashi, K., Masuda, A., Tokumoto, M., Ozaki, Y., Saito, K., ... Sakai, N. (2013). Properties of gene knockdown system by vector-based siRNA in zebrafish. Development Growth and Differentiation, 55(9), 755-765. https://doi.org/10.1111/dgd.12091

Properties of gene knockdown system by vector-based siRNA in zebrafish. / Shinya, Minori; Kobayashi, Kayo; Masuda, Aki; Tokumoto, Mika; Ozaki, Yuichi; Saito, Kenji; Kawasaki, Toshihiro; Sado, Yukiko; Sakai, Noriyoshi.

In: Development Growth and Differentiation, Vol. 55, No. 9, 12.2013, p. 755-765.

Research output: Contribution to journalArticle

Shinya, M, Kobayashi, K, Masuda, A, Tokumoto, M, Ozaki, Y, Saito, K, Kawasaki, T, Sado, Y & Sakai, N 2013, 'Properties of gene knockdown system by vector-based siRNA in zebrafish', Development Growth and Differentiation, vol. 55, no. 9, pp. 755-765. https://doi.org/10.1111/dgd.12091
Shinya, Minori ; Kobayashi, Kayo ; Masuda, Aki ; Tokumoto, Mika ; Ozaki, Yuichi ; Saito, Kenji ; Kawasaki, Toshihiro ; Sado, Yukiko ; Sakai, Noriyoshi. / Properties of gene knockdown system by vector-based siRNA in zebrafish. In: Development Growth and Differentiation. 2013 ; Vol. 55, No. 9. pp. 755-765.
@article{f8465b636b2f40fb9539bc761dc54064,
title = "Properties of gene knockdown system by vector-based siRNA in zebrafish",
abstract = "RNA interference (RNAi) has emerged as a powerful tool to silence specific genes. Vector-based RNAi systems have been developed to downregulate targeted genes in a spatially and temporally regulated fashion both in vitro and in vivo. The zebrafish (Danio rerio) is a model animal that has been examined based on a wide variety of biological techniques, including embryonic manipulations, forward and reverse genetics, and molecular biology. However, a heritable and tissue-specific knockdown of gene expression has not yet been developed in zebrafish. We examined two types of vector, which produce small interfering RNA (siRNA), the direct effector in RNAi system; microRNA (miRNA) process mimicking vectors with a promoter for RNA polymerase II and short hairpin RNA (shRNA) expressing vector through a promoter for RNA polymerase III. Though gene-silencing phenotypes were not observed in the miRNA process mimicking vectors, the transgenic embryos of the second vector (Tg(zU6-shGFP)), shRNA expressing vector for enhanced green fluorescence protein, revealed knockdown of the targeted gene. Interestingly, only the embryos from Tg(zU6-shGFP) female but not from the male fish showed the downregulation. Comparison of the quantity of siRNA produced by each vector indicates that the vectors tested here induced siRNA, but at low levels barely sufficient to silence the targeted gene.",
keywords = "RNAi, ShRNA, SiRNA, U6 snRNA promoter",
author = "Minori Shinya and Kayo Kobayashi and Aki Masuda and Mika Tokumoto and Yuichi Ozaki and Kenji Saito and Toshihiro Kawasaki and Yukiko Sado and Noriyoshi Sakai",
year = "2013",
month = "12",
doi = "10.1111/dgd.12091",
language = "English",
volume = "55",
pages = "755--765",
journal = "Development, growth & differentiation",
issn = "0012-1592",
publisher = "Wiley-Blackwell",
number = "9",

}

TY - JOUR

T1 - Properties of gene knockdown system by vector-based siRNA in zebrafish

AU - Shinya, Minori

AU - Kobayashi, Kayo

AU - Masuda, Aki

AU - Tokumoto, Mika

AU - Ozaki, Yuichi

AU - Saito, Kenji

AU - Kawasaki, Toshihiro

AU - Sado, Yukiko

AU - Sakai, Noriyoshi

PY - 2013/12

Y1 - 2013/12

N2 - RNA interference (RNAi) has emerged as a powerful tool to silence specific genes. Vector-based RNAi systems have been developed to downregulate targeted genes in a spatially and temporally regulated fashion both in vitro and in vivo. The zebrafish (Danio rerio) is a model animal that has been examined based on a wide variety of biological techniques, including embryonic manipulations, forward and reverse genetics, and molecular biology. However, a heritable and tissue-specific knockdown of gene expression has not yet been developed in zebrafish. We examined two types of vector, which produce small interfering RNA (siRNA), the direct effector in RNAi system; microRNA (miRNA) process mimicking vectors with a promoter for RNA polymerase II and short hairpin RNA (shRNA) expressing vector through a promoter for RNA polymerase III. Though gene-silencing phenotypes were not observed in the miRNA process mimicking vectors, the transgenic embryos of the second vector (Tg(zU6-shGFP)), shRNA expressing vector for enhanced green fluorescence protein, revealed knockdown of the targeted gene. Interestingly, only the embryos from Tg(zU6-shGFP) female but not from the male fish showed the downregulation. Comparison of the quantity of siRNA produced by each vector indicates that the vectors tested here induced siRNA, but at low levels barely sufficient to silence the targeted gene.

AB - RNA interference (RNAi) has emerged as a powerful tool to silence specific genes. Vector-based RNAi systems have been developed to downregulate targeted genes in a spatially and temporally regulated fashion both in vitro and in vivo. The zebrafish (Danio rerio) is a model animal that has been examined based on a wide variety of biological techniques, including embryonic manipulations, forward and reverse genetics, and molecular biology. However, a heritable and tissue-specific knockdown of gene expression has not yet been developed in zebrafish. We examined two types of vector, which produce small interfering RNA (siRNA), the direct effector in RNAi system; microRNA (miRNA) process mimicking vectors with a promoter for RNA polymerase II and short hairpin RNA (shRNA) expressing vector through a promoter for RNA polymerase III. Though gene-silencing phenotypes were not observed in the miRNA process mimicking vectors, the transgenic embryos of the second vector (Tg(zU6-shGFP)), shRNA expressing vector for enhanced green fluorescence protein, revealed knockdown of the targeted gene. Interestingly, only the embryos from Tg(zU6-shGFP) female but not from the male fish showed the downregulation. Comparison of the quantity of siRNA produced by each vector indicates that the vectors tested here induced siRNA, but at low levels barely sufficient to silence the targeted gene.

KW - RNAi

KW - ShRNA

KW - SiRNA

KW - U6 snRNA promoter

UR - http://www.scopus.com/inward/record.url?scp=84890792973&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84890792973&partnerID=8YFLogxK

U2 - 10.1111/dgd.12091

DO - 10.1111/dgd.12091

M3 - Article

C2 - 24117364

AN - SCOPUS:84890792973

VL - 55

SP - 755

EP - 765

JO - Development, growth & differentiation

JF - Development, growth & differentiation

SN - 0012-1592

IS - 9

ER -