A robust system for RNA interference in the chicken using a modified microRNA operon.
RNA interference (RNAi) provides an effective method to silence gene expression and investigate gene function. However, RNAi tools for the chicken embryo have largely been adapted from vectors designed for mammalian cells. Here we present plasmid and retroviral RNAi vectors specifically designed for optimal gene silencing in chicken cells. The vectors use a chicken U6 promoter to express RNAs modelled on microRNA30, which are embedded within chicken microRNA operon sequences to ensure optimal Drosha and Dicer processing of transcripts. The chicken U6 promoter works significantly better than promoters of mammalian origin and in combination with a microRNA operon expression cassette (MOEC), achieves up to 90% silencing of target genes. By using a MOEC, we show that it is also possible to simultaneously silence two genes with a single vector. The vectors express either RFP or GFP markers, allowing simple in vivo tracking of vector delivery. Using these plasmids, we demonstrate effective silencing of Pax3, Pax6, Nkx2.1, Nkx2.2, Notch1 and Shh in discrete regions of the chicken embryonic nervous system. The efficiency and ease of use of this RNAi system paves the way for large-scale genetic screens in the chicken embryo.
Das
Raman M
RM
Department of Molecular Biology and Biotechnology, The University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK.
Van Hateren
Nick J
NJ
Howell
Gareth R
GR
Farrell
Elizabeth R
ER
Bangs
Fiona K
FK
Porteous
Victoria C
VC
Manning
Elizabeth M
EM
McGrew
Michael J
MJ
Ohyama
Kyoji
K
Sacco
Melanie A
MA
Halley
Pam A
PA
Sang
Helen M
HM
Storey
Kate G
KG
Placzek
Marysia
M
Tickle
Cheryll
C
Nair
Venugopal K
VK
Wilson
Stuart A
SA
D000818:Animals; D002460:Cell Line; D002642:Chick Embryo / Q000033:anatomy & histology / Q000502:physiology; D018507:Gene Expression Regulation, Developmental; D020868:Gene Silencing; D005822:Genetic Vectors; D000090322:Homeobox Protein Nkx-2.2; D018398:Homeodomain Proteins; D006801:Humans; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins; D009876:Operon; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D051881:Receptor, Notch1 / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors
D000090322:Homeobox Protein Nkx-2.2; D018398:Homeodomain Proteins; D035683:MicroRNAs; C000715631:NKX2-2 protein, human; D009687:Nuclear Proteins; D051881:Receptor, Notch1; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors