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4,5to
their widespread use, there are
substantial limitations. These
include the fact that the multiple
copies of the experimental gene will
express in unpredictable—
frequently excessive—amounts.
Additionally, expression from the
transgenes can be silenced or
induce cosuppression (silencing of
homologous gene sequences on the
chromosome), especially in the
germline
C. elegans select
Helen M Chamberlin
The technical toolkit for Caenorhabditis elegans
expands to include experimental selection using
antibiotic resistance genes.
3In
C. elegans, transgenes can be
rapidly produced by injection of
experimental DNA (such as plasmid
DNA) directly into the gonad of a
parental worm. In the offspring, the
injected DNA is assembled into large,
extrachromosomal arrays by
recombination. Conveniently, the
transgenic arrays can incorporate
representatives of any DNAs included
in an injected mixture, bypassing the
need to covalently link the
experimental DNA to other reporter
DNA included as a transformation
positive control. These arrays are
replicated and transmitted through
mitosis and meiosis in a semistable
manner in the worm, even without the
inclusion of chromosomal features
such as telomere or centromere
sequences in the injected DNA.
cells to produce stably transmitted
Although the relative ease with
transgenes is an essential tool to
which these C. elegans multicopy
incorporate new or modified genes into extrachromosomal transgenes can
research organisms or to otherwise
be produced has led
manipulate their genomes.
1Genetic
selection systems that use
antibiotics in combination with
antibiotic resistance genes are the
workhorse of any molecular biology
laboratory. These flexible tools allow
stringent, conditional selection of
experimentally manipulated
individuals. Until now, however, their
broad application has been limited to
studies in singlecell organisms,
cultured cells and plants. New work
reported in Semple et al. 2 and
Giordano-Santini et al.1,2 in this issue of
Nature Methods applies these
selection systems to C. elegans and
related nematodes, adding this
powerful method to the C. elegans
molecular biology toolkit. Introduction
of experimental DNA into
Transposon
systems • DNA
microinjection •
DNA
electroporation
Identify a DNA
transformation
strategy
• Viral vectors •
Animal transgenesis
system
6.
Furthermore, injected marker
systems used to identify the
transgenes can be identified
visually but generally do not
provide ‘selection’ strategies in
which non–transgene-bearing
worms do not survive. When
selection is incorporated, the
system requires introduction of
specific C. elegans mutations into
the genetic background of an
experimental strain, limiting the use
of such strategies in large-scale
experiments and in non– C.
elegans nematodes. Some of these
shortcomings have been recently
addressed by the optimization of
methodology for integration of a
singlecopy transgene into a
chromosomal locus, using a
transposon excision and repair
strategy1. Although this method
addresses the problems of
transgene expression amount and
silencing that arise from multiple
gene copies, it is still constrained by
the selection marker systems
available for C. elegans. Enter
Semple et al.2 and
GiordanoSantini et al.
Develop antibiotic resistance gene vector
1, to provide the missin
• Species-specific ubiquitous promoter
puzzle. These groups
• Antibiotic resistance gene
antibiotic resistance ge
are widely used in euk
(such as yeast and cul
expand the transgenic
systems available for C
show that worms with
expression of antibiotic
genes (PuroR or NeoR
and that the gene expr
resistance to drug con
(puromycin or G-418),
otherwise kill the worm
Semple et al.
Define selection
conditions
Figure 1 | Developing animal transgenesis methods. The selection approach of Semple et
al.12 and Giordano-Santini et al. combined with identification of species-appropriate DNA
transformation strategies yields a general scheme to develop a transgenic system for
experimental animals.
Helen M. Chamberlin is at the Department
of Molecular Genetics, Ohio State University, Columbus, Ohio, USA. e-mail:
chamberlin.27@osu.edu
bypass a problem encountered in
the past by other researchers (the
relative inaccessibility of cells in intact
worms to externally administered
antibiotic, necessitating large
treatment doses) by including small
amounts of detergent when they
subjected worms to drug selection.
Both groups also show that their
selection systems are
© 2010 Nature America, Inc. All rights reserved.
nature methods | VOL.7 NO.9 | SEPTEMBER 2010 |
693
~10× more fat cells
Add soluble factorsSoft and thick like fat ~2× more bone
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MSC
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difference with flexible substrates has been
observed to influence the differentiation of
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Methods, Chen and co-workers have made
dense arrays of various-flexibility pillars for
cells to adhere on top of and show that the
potency of soluble factors that induce either
fat or bone depends on pillar flexibility
Figure 1 | Adherent stem
to a solid substrate to sur
the cells attach to a subst
one that is stiff can influe
Amnon Buxboim and De
the University of Pennsy
Pennsylvania, USA. e-m
discher@seas.upenn.ed
1.Fat
is
softe
r
than
bone
, and
mimi
cking
this
tebrate experimental animals (Fig. 1).
The use of an antibiotic resistance
system means that a detailed genetic
analysis is not required before
developing transgenesis methods for
an experimental species.
Consequently, the work uncovers a
longterm opportunity for pan-species
transgenesis in comparative studies of
gene function and gene evolution.
i
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ComPetinG FinanCiaL interests The author
declares no competing financial interests.
1. Semple, J. et al. Nat. Methods. 7, 725–727
(2010). 2. Giordano-Santini, R. et al. Nat.
Methods. 7,
721–723 (2010). 3. Mello, C.C., Kramer,
J.M., Stinchcomb, D. &
Ambros, V. EMBO J. 10, 3959–3970 (1991).
4. Kelly, W.G., Xu, S., Montgomery, M.K. & Fire,
A.
Genetics 146, 227–238 (1997). 5.
Dernburg, A.F., Zalevsky, J., Colaiácovo, M.P.
& Villeneuve, A.M. Genes Dev. 14,
1578–1583 (2000).
6. Frøkjaer-Jensen, C. et al. Nat. Genet. 40, 1375–1383 (2008).
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Katie Vicari
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responsive with a si
antibiotic resistance
with the transposon
and-repair method.
streamlined single-c
transgene system th
the strict conditional
antibiotic selection.
2The benefits of the
antibiotic selection s
beyond the single-c
application. Indeed,
systems are used a
maintain traditional
extrachromosomal t
result in populations
retain the transgene
100% frequency, im
reproducibility and c
experiments carrie
traditional transgene
also be used with o
species that lack the
toolbox of C. elegan
show the selection s
the related C. briggs
Giordano-Santini et
G-418 sensitivity in
nematode species.