Transgenesis in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 162

be capable of generating transgenes in C. elegans, some of which integrate randomly
into the genome at low or even single copy ( Praitis et al., 2001 ). These types of
transgenes generally overcome the limitations of high-copy arrays and are able to
express more efficiently in the germ line.
What has lagged behind in the C. elegans field is a robust method for single-copy
gene insertions and targeted chromosomal modifications. Such modifications
would, by their nature, permit expression of maternal and zygotic genes under the
control of endogenous regulatory elements and allow generation of custom-made
alleles.
Two general methods to generate homologous recombinants, both of which
depend on either microinjection or microparticle bombardment to generate trans-
genic lines, have been developed in the last few years ( Fig. 1C ). One approach takes
advantage of the excision of a transposable element to create a double-stranded (ds)
break in DNA, which can be used to promote gene conversion or direct insertion of
transgenic sequences directly into the chromosome ( Frokjaer-Jensen et al., 2008;
Plasterk and Groenen, 1992; Robert et al.,2009 ). Genomewide screens that have
produced thousands of Tc1 and Mos1 transposon insertion lines have significantly
increased the applicability of this approach ( Bazopoulou and Tavernarakis, 2009;
Boulin and Bessereau, 2007; Duverger et al., 2007; Williams et al.,2005 ). In a
second approach, scaled-up methods for microparticle bombardment have been
used to produce integrations targeted at the endogenous locus ( Berezikov et al.,
2004 ) and recent work using a positive- and negative-selection strategy promises to
dramatically improve the efficiency of this process ( Vazquez-Manrique

et al.,

2010 ).
Comprehensive protocols for generating transgenics by microinjection and micro-
particle bombardment are available online, in the WormMethods section of
WormBook and in a variety of other excellent published sources ( Evans, 2006;
Green et al., 2008; Hope, 1999; Kadandale et al., 2009; Mello and Fire, 1995;
Praitis, 2006; Praitis et al., 2001; Rieckher et al., 2009 ). What follows are brief
descriptions of the uses of transgenes in C. elegans research, general considerations
for constructing transgenes and delivering them to C. elegans, an assessment of
methods in other nematodes, and a brief discussion of what future developments may
lie ahead.

II. Uses for Transgenes in C. elegans

A. Analysis of Gene Expression
The most frequent use of transgenes in C. elegans is for the assessment of
endogenous gene-expression patterns of protein-coding genes. The simplest
approach for making a transcriptional reporter is to clone the 5 0 regulatory sequence
from a gene of interest, fuse it to a reporter gene whose activity can be easily assayed,
and include a 3 0 UTR, usually that of the unc-54 gene ( Fire et al., 1990 ). Because of