Article: Take out the trash
- From: "Robert Karl Stonjek" <rstonjek@xxxxxxxxxxxxxx>
- Date: Fri, 3 Aug 2007 20:02:57 -0400 (EDT)
Take out the trash
Neuroscience Gateway (July 2007) | doi:10.1038/aba1764
StandfirstA new technique deletes large noncoding regions of the mouse
genome.
One man's junk is another man's treasure. Several international
collaborations plan to knock out every mouse gene, but gene-coding regions
only constitute approximately 2.5% of the genome. What about the remaining
97.5% of the genome containing so-called 'junk DNA'? Wu et al. report a
high-throughput method to efficiently knock out large genetic regions in the
mouse in a recent article in Nature Genetics.
Cre-loxP recombination is commonly used to generate knockout mice.
Researchers insert loxP sites into genetic regions flanking the gene they
want to knock down. They mate mice with the 'floxed' allele to mice
expressing Cre recombinase, which excises the sequence between loxP
recognition sites in resulting progeny, leaving behind one loxP tag.
Can Cre excise genetic regions between loxP sites located on two different
alleles? A total of 58 genes encoding the membrane adhesion molecules
protocadherins (Pcdh) are sequentially located in three clusters (, and )
in a 1 Mb region on mouse chromosome 18. The authors bred mice with
homozygous Cre-loxP-mediated mutations of different Pcdh genes in the same
or different Pcdh gene clusters. Some of the resulting progeny carried loxP
sites in different regions of Pcdh on each allele. The authors bred these
mice with Cre-expressing mice and found both deletions and duplications of
the sequences between trans-allelic loxP sites. Mice bred from lines with
loxP sites separated by the shortest distance (54 kb) showed the highest
recombination frequency (21.6%). Approximately 9.3% of mice bred from lines
with loxP sites in and clusters had 700 kb Pcdh deletions and
duplications, suggesting that Cre can mutate genetic regions located between
trans-allelic loxP sites separated by relatively large genetic distances.
When the authors bred mice with loxP sites on different chromosomes, they
found a small number of progeny with chromosomal translocations.
To efficiently scatter loxP sites throughout the genome, they modified a
Drosophila genetics tool. The piggyBac transposon inserts randomly into TTAA
sequences in the target genome. Traditionally, piggyBac is used in gene-trap
experiments, which simultaneously report where a gene or enhancer is
expressed and disrupt its normal expression. The authors designed a
gene-trap vector with two reporters, beta-galactosidase (-gal) and green
fluorescent protein (GFP), as well as loxP and Flp recombinase FRT sites.
They injected mouse embryos with gene-trap vectors and piggyBac transposase,
which catalyzes transposon insertion. Mice carrying the piggyBac vector were
easily selected because they fluoresced. The authors bred Cre-expressing
mice with mice carrying piggyBac gene-trap alleles in Mdc1 and Runx2, which
are separated by 8.6 Mb on chromosome 17, and found mice with deletions and
duplications of the intervening sequence among the progeny.
Therefore, Cre-mediated recombination of piggyBac-inserted loxP sites
mutates large regions of the genome. Phenotype screens in mice with random
mutations might identify functional roles for junk DNA in normal physiology
and behavior.
Author: Debra Speert
Wu, S., Ying, G., Wu, Q. & Capecchi, M. R. Toward simpler and faster
genome-wide mutagenesis in mice. Nature Genetics 39, 922-930 (2007). |
Article |
Source: Nature Neuroscience
http://www.brainatlas.org/aba/2007/070726/full/aba1764.shtml
Posted by
Robert Karl Stonjek
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