Biology Reference
In-Depth Information
export/influx/efflux and transport, cell division, morphology, as well as circadian
rhythms (
Flockhart et al. 2006, Mohr et al. 2010
). In addition, the center provides
information on reagents, database and software tools for analysis of data, and
the off-target effects of RNAi. RNAi using
Drosophila
can be conducted using
high-throughput methods, which allows diverse processes to be studied and
identification of new components of physiological processes (
Mohr et al. 2010
).
Mohr et al. (2010)
concluded RNAi is “a powerful method for genome-scale
interrogation of gene function
…
” but “The results of RNAi high-throughput
screens (HTSs) are acutely sensitive to assay design and are subject to significant
rates of false discovery, which can be addressed using various statistical, bioin-
formatics and experimental approaches.”
Mohr et al. (2010)
also highlighted
the need for improved methods, standardization of data reporting, and other
methodological improvements.
Unfortunately, feeding ds RNA to
D. melanogaster
does not appear to work,
as it does in
C. elegans
. RNAi reagents must be delivered by injection into pre-
cellular-blastoderm embryos or as transgenes. RNAi analyses using injection of
embryos limits the study of gene function to those genes active during embry-
onic development. Transgenic RNAi can be used to study gene function in
somatic tissues, but the RNAi is cell-autonomous in
D. melanogaster
. So far, RNAi
works well in somatic tissues, but does not work in the female germ line, for
unknown reasons (
Perrimon et al. 2010, Ni et al. 2011
). However,
Ni et al. (2011)
are constructing RNAi tools for all 14,208 protein-coding genes in
Drosophila
so
that the
Drosophila
community can conduct RNAi studies on these genes.
RNAi also is being studied in other insects and is considered a possible tool for
obtaining control of pests (see Chapter 14).
9.19.3 Zinc-Finger Nucleases (ZFNs)
Zinc-finger nucleases (ZFNs) are artificial molecules composed of a
Fok
I type-II
restriction endonuclease and zinc fingers. ZFNs are being studied as a mechanism
to genetically modify arthropods without using TE vectors. ZFNs can be used to
create targeted double-stranded breaks in the genome that result in targeted
mutagenesis or in gene replacement (
Kim et al. 1996, Carroll 2011
). The enzymes
consist of a DNA-binding region, which can be designed to bind to a particular
sequence in the genome, and a
Fok
I endonuclease domain, which introduces the
break (
Carroll 2011; deSouza 2011, 2012; Baker 2012
). The binding domain con-
sists of a tandem array of Cys
2
His
2
zinc fingers, each of which recognizes three
nucleotides in the target DNA sequence. By linking together multiple fingers
(three to six), ZFN pairs can be designed to bind to genomic sequences 18-36nt
long. Each zinc finger contacts 3bp of DNA through interactions in the major
groove of the DNA and fingers that recognize many of the 64 triplets have been
