Testing the efficiency of dsRNAi constructs in vivo: a transient expression assay based on two fluorescent proteins.
Double-stranded RNA interference technology has recently been shown to be a powerful tool to silence gene expression in various organisms, including plants. Sustained double-stranded RNA interference mediated gene silencing is normally triggered by hairpin RNAs generated by in vivo transcription of inverted repeat DNA constructs. To test the efficiency of inverted repeat constructs for their in vivo gene silencing capability, we developed an assay that is based on transient expression in single cells using two fluorescent reporter proteins with different emission spectra. We co-expressed one fluorescent protein as marker for transfected cells and the second as translational fusion with the target gene. Co-transfection of a vector mediating expression of inverted repeats of the target gene resulted in a specific decrease or undetectable fusion protein fluorescence in the majority of transfected cells.
Panstruga
Ralph
R
Max-Planck-Institut für Züchtungsforschung, Department of Plant Microbe Interactions, Carl-von-Linné-Weg 10, 50829 Köln, Germany. panstrug@mpiz-koeln.mpg.de
Kim
Min Chul
MC
Cho
Moo Je
MJ
Schulze-Lefert
Paul
P
D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins; D001467:Hordeum / Q000166:cytology / Q000235:genetics* / Q000378:metabolism; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000235:genetics* / Q000378:metabolism*; D000097573:Red Fluorescent Protein
D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D012330:RNA, Double-Stranded; D000097573:Red Fluorescent Protein