A pipeline for the generation of shRNA transgenic mice.
RNA interference (RNAi) is an extremely effective tool for studying gene function in almost all metazoan and eukaryotic model systems. RNAi in mice, through the expression of short hairpin RNAs (shRNAs), offers something not easily achieved with traditional genetic approaches-inducible and reversible gene silencing. However, technical variability associated with the production of shRNA transgenic strains has so far limited their widespread use. Here we describe a pipeline for the generation of miR30-based shRNA transgenic mice that enables efficient and consistent targeting of doxycycline-regulated, fluorescence-linked shRNAs to the Col1a1 locus. Notably, the protocol details crucial steps in the design and testing of miR30-based shRNAs to maximize the potential for developing effective transgenic strains. In all, this 14-week procedure provides a fast and cost-effective way for any laboratory to investigate gene function in vivo in the mouse.
Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA.
Lukas E
LE
Dow
Prem K
PK
Premsrirut
Johannes
J
Zuber
Christof
C
Fellmann
Katherine
K
McJunkin
Cornelius
C
Miething
Youngkyu
Y
Park
Ross A
RA
Dickins
Gregory J
GJ
Hannon
Scott W
SW
Lowe
D000818:Animals; D003001:Cloning, Molecular; D024042:Collagen Type I / Q000235:genetics; D000090602:Collagen Type I, alpha 1 Chain; D005818:Genetic Engineering / Q000379:methods*; D060005:Genotyping Techniques; D051379:Mice; D008822:Mice, Transgenic; D035683:MicroRNAs / Q000235:genetics; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*
D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D035683:MicroRNAs; C534715:Mirn30d microRNA, mouse; D034741:RNA, Small Interfering