Questions & Answers
About ddRNAi Expression Vectors
What is a ddRNAi expression vector? ddRNAi expression vectors rely on RNA polymerase III-promoters (e.g. U6 or H1) for the expression of siRNA target sequences transfected in mammailain cells. siRNA target sequences generated from
ddRNAi expression cassette system can be directly cloned into a vector that does not contain a U6 promoter. Alternatively short single stranded DNA oligos containing the hairpin siRNA target sequence are annealed and cloned into a vector downsteam of the pol III promoter.
What is a hairpin? Most strategies for cloning siRNA target sequences into expression vectors utilize the design of a hairpin structure. This design consists of two inverted repeats separated by
a short spacer sequence (loop sequence). After transcription by polymerase III, the inverted repeats anneal and form
a hairpin.
What is the primary advantage of using ddRNAi
expression vectors? The primary advantage of ddRNAi expression vectors is that they allow for long term interference experiments. Vectors with antibiotic markers such as
puromycin, neomycin or hygromycin can be used for suppression of target genes for several weeks or longer. Transfection with synthetic siRNAs allows for only a transient measurement
(usally 48-72 hours) of the RNAi effect.
What does the presence of antibiotic selection allow? The presence of antibiotic selection marker has several advantages:
A) Antibiotic markers allow for the selection of only cells that have been successfully transfected. If the transfection efficiency is low only a fraction of the cells will contain the plasmid that will express the siRNA target sequence. Detection of suppression (such as Western blot using a crude cell lysate) would be diluted and possibly be observed as a false negative. Using a homogeneous population of cells that contains the plasmid that will express the siRNA target sequence compensates for low transfection efficiency and allows for the detection of minimal interference levels.
B) Since synthetic siRNAs allow for only transient affect (2-3 days), phenotypic changes correlating with proteins with long half lifes would be overlooked. Antibiotic selection enables the RNA interference experiments to be carried out for several weeks to months.
C) The
use of most transfection reagents has some negative effect on cell physiology
and proliferation. Allowing cells (post transfection) time to return to a normal
physiological state would minimize any non-specific RNA interference that is related to the transfection procedure.
For a general review of ddRNAi expression vectors
consult these references:
