Biology Reference
In-Depth Information
Box 9.2 Some Newer Tools for Modifying the Genome of
Drosophila
and Other Insects
Homing endonucleases (HEs) or meganucleases
Homing nucleases, or meganuleases, are sequence-specific endonucleases with large cleavage sites
(14-25 bp) that can create double-stranded breaks at specific locations in the genome. Meganucleases
are encoded by mobile genetic elements that induce recombination in a process called homing. Several
hundred naturally occurring HEs have been identified but the likelihood of finding a HE cleavage site in a
particular gene can be low. Artificial meganucleases now can be produced with a specificity determined by
the target gene (
Smith et al. 2006, Fajardo-Sanchez 2008, Baker 2012, deSouza 2012
).
Transcription activator-like effector nucleases (TALENS)
TALENs are artificial molecules that have a DNA-binding domain from TAL effector proteins, which are
transcription factors from plant-pathogenic bacteria (
Xanthomonas
), and the
Fok
I endonuclease. THE
DNA-binding domain is a tandem array of amino-acid repeats, each
≈
34 residues long. Each TALEN
repeat binds to a single base pair. TALENs cleave DNA, have relatively long target sequences and are
perhaps less difficult to use than ZFNs (
Baker 2012, de Souza 2012
).
Zinc-finger nucleases (ZFNs)
ZFNs are synthetic molecules containing three or four zinc fingers linked to the
Fok
I nuclease domain
that can be used as gene-targeting tools. ZFNs induce double-stranded breaks that result in DNA repair
processes that can produce both targeted mutagenesis and targeted gene replacement. ZFNs must
be developed for each genomic target. The new DNA (exogenous gene) is inserted by homologous
recombination between the original and exogenous gene copies. First, specific DNA sequences are cleaved
by the ZFN, donor DNA is added, and homologous recombination will result in gene replacement. Or,
nonhomologous end-joining can result in targeted mutagenesis, resulting in deletions, insertions or base
substitutions. Because the cleavage domain has no specificity, recognition domains can be inserted that
recognize specific domains that should result in targeted cleavages (
Carroll 2011, Baker 2012, Isalan
2012
). Unfortunately, a substantial proportion of ZFNs fail, so multiple sequences must be tested for a
single target gene to identify the most effective. Nontarget cleavages also can occur so extensive tests are
conducted to confirm efficacy. ZFNs are commercially available because they are difficult for the average
research group to build on their own.
9.19.1 JcDNV Gene Vectors for Somatic Transformations
A lepidopteran densovirus isolated from
Junonia coenia
(JcDNV) and modified
to serve as a vector produces somatic transformations that can be used to exam-
ine the regulation of transgenes in insects (
Bossin et al. 2007
). Microinjection of
these plasmids into pre-blastoderm embryos of
D. melanogaster
,
Plodia inter-
punctella
,
Ephestia kuehniella
, and
Trichoplusia ni
allowed transgene expression
throughout development and evaluation of different tissue-specific promoters.
9.19.2 RNAi for
Drosophila
RNA interference (RNAi) is an evolutionarily conserved cellular defense mecha-
nism for controlling the expression of alien DNA in plants and animals. RNAi
also is involved in controlling development.
Fire et al. (1998)
discovered RNAi
when the application of exogenous ds RNA silenced the homologous mRNA
