Specialized Chromosomes and Their Uses in Caenorhabditis elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 49

straightforward approach for generating a wild-type clone is to PCR the sequence
directly from genomic DNA. This approach requires the use of a high-fidelity poly-
merase and that the region to be amplified is of a suitable size for reliable PCR
amplification.

2. Expression Pattern Analysis
The transparency and size of C. elegans allows for visualization of gene expres-
sion patterns using the reporter genes in vivo without animal dissection. The E. coli
gene LacZ, encoding b -galactosidase, was first used for expression analysis in
C. elegans ( Fire et al., 1990 ) but has since been superseded with the introduction
of GFP, the use of which was pioneered in this organism ( Chalfie et al., 1994 ).
Expression reporters can be transcriptional or translational. Transcriptional reporter
construct contain the native promoter elements of the gene of interest which are used
to drive expression of the reporter. Generally, making transcriptional reporters is
straightforward and uses cloning methods or simple and efficient PCR-based meth-
ods ( Hobert, 2002; Hunt-Newbury et al., 2007; McKay et al., 2003 ). Alternatively,
translational reporters can be constructed where the reporter is fused into the protein-
coding sequence providing a hybrid protein. Translational reporters often faithfully
replicate the native gene ' s function though using this approach may require that
several constructs be created to find a position in the gene where the insertion of GFP
does not inhibit the function of the native protein ( Merritt and Seydoux, 2010 ).
Generally, using the translational reporter to rescue the phenotype of a mutation is
sufficient to confirm that all promoter elements are present and that the hybrid
protein functions in the same way as the native protein.

3. Ectopic Expression Reporters
For some purposes it is not necessary, or even desirable, to use the endogenous
promoter. A variety of endogenous promoter elements have been characterized in
C. elegans ( Davis et al., 2008 ). Such promoters can be used to drive ectopic
expression of a gene of interest in a known tissue type or temporal pattern ( Kalb
et al., 1998; Mah et al., 2007; McGhee et al., 2009 ). As more promoter elements are
discerned more become available and an effort to fully characterize tissue-specific
promoters is underway (M. Chalfie pers. comm.). Additionally, conditional promo-
ters are available for inducing gene expression under a variety of conditions includ-
ing various heat-shock elements ( Seydoux et al., 1996 ).

4. Other Uses

Transgenic arrays are not limited to gene expression studies but have also been
used to create a plethora of experimental tools. Some of the many examples include:
tissue-specific RNAi analysis ( Briese et al., 2006; Qadota et al., 2007; Voutev and
Hubbard, 2008 ); identification of synaptic partners in the nervous system