Biology Reference
In-Depth Information
5. Single-Gene Deletions
Single-gene deletions, often referred to as gene knockouts, are valuable tools for
determining gene function. Knockouts are generated using mutagens that create
small deletions, most commonly trimethyl psoralen (TMP) followed by UV-radiation
(UV-TMP). In C. elegans, there is an international effort to generate gene knockouts
for the research community (
Barstead and Moerman, 2006; Mitani, 2009; Moerman
and Barstead, 2008
). Many knockout strains are available from the Caenorhabditis
Genetics Center (CGC) (
www.cbs.umnedu/CGC/strains/
) and are also listed in
Wo rmBa s e (
www.wormbase.org
). Requests to have a knockout allele generated
for a specific gene should be sent to either the National Bioresource Project,
shigen.nig.ac.jp/c.elegans/index.jsp
], or The Gene Knockout Project, University
6. Advantages and Disadvantages
Mapping using Dfs has the advantage of being able to position a mutation of
interest to a region of the genome with higher resolution than can be done using more
traditional genetic approaches such as three-factor mapping. However, deletions can
be complex and not delete contiguous stretches of DNA. Without the aid of cytology
to characterize deletions, most deletions have not been described molecularly reduc-
ing the mapping resolution with regard to the DNA sequence.
Many deletions disrupt at least one essential gene and are therefore lethal. In fact,
they often affect more than one essential gene and are arrested during the embryo
stage when homozygous. Thus, they, as for any lethal mutation, need to be maintained
using a balancer. This is however normally straightforward. Single-gene deletions
greatly facilitate correlating phenotype with genotype and provide a substrate for
molecular PCR-based assays to follow phenotype. Mutations that are phenotypically
null are critical for interpretation of gene function. Single-gene knockout are impor-
tant because they are more likely to be null for gene function than point mutations that
can often be hypomorphic. Experimentally, they are relatively easy to use and genes
that do not display a viable visible phenotype can be followed using a simple PCR
assay to determine if the strain contains the mutation either homozygously or hetero-
zygously. A special subset of deletions reduce crossing over in adjacent regions and
these can, in principal, be used to balance lethals on the homolog.
D. Duplications
Genomic duplications have proven useful for a number of applications in C.
elegans research. In general, two types of duplications are available, ones that are
inserted into a chromosome and ones that exist as extrachromosomal ''free'' ele-
ments. Duplications that are inserted into the genome segregate in a Mendelian
fashion and are stably maintained. Free duplications generally exist as single copies,
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