A Drosophila resource of transgenic RNAi lines for neurogenetics.
Conditional expression of hairpin constructs in Drosophila is a powerful method to disrupt the activity of single genes with a spatial and temporal resolution that is impossible, or exceedingly difficult, using classical genetic methods. We previously described a method (Ni et al. 2008) whereby RNAi constructs are targeted into the genome by the phiC31-mediated integration approach using Vermilion-AttB-Loxp-Intron-UAS-MCS (VALIUM), a vector that contains vermilion as a selectable marker, an attB sequence to allow for phiC31-targeted integration at genomic attP landing sites, two pentamers of UAS, the hsp70 core promoter, a multiple cloning site, and two introns. As the level of gene activity knockdown associated with transgenic RNAi depends on the level of expression of the hairpin constructs, we generated a number of derivatives of our initial vector, called the "VALIUM" series, to improve the efficiency of the method. Here, we report the results from the systematic analysis of these derivatives and characterize VALIUM10 as the most optimal vector of this series. A critical feature of VALIUM10 is the presence of gypsy insulator sequences that boost dramatically the level of knockdown. We document the efficacy of VALIUM as a vector to analyze the phenotype of genes expressed in the nervous system and have generated a library of 2282 constructs targeting 2043 genes that will be particularly useful for studies of the nervous system as they target, in particular, transcription factors, ion channels, and transporters.
Ni
Jian-Quan
JQ
Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, Virginia 20147, USA.
Liu
Lu-Ping
LP
Binari
Richard
R
Hardy
Robert
R
Shim
Hye-Seok
HS
Cavallaro
Amanda
A
Booker
Matthew
M
Pfeiffer
Barret D
BD
Markstein
Michele
M
Wang
Hui
H
Villalta
Christians
C
Laverty
Todd R
TR
Perkins
Lizabeth A
LA
Perrimon
Norbert
N
D000818:Animals; D002352:Carrier Proteins / Q000235:genetics; D004330:Drosophila / Q000235:genetics*; D055785:Gene Knockdown Techniques / Q000379:methods*; D007473:Ion Channels / Q000235:genetics; D008722:Methods; D009420:Nervous System; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics
D002352:Carrier Proteins; D007473:Ion Channels; D034741:RNA, Small Interfering; D014157:Transcription Factors