Specialized Chromosomes and Their Uses in Caenorhabditis elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 50

( Feinberg et al., 2008 ); investigation of meiotic processes ( Mets and Meyer, 2009 );
and investigation of endosomal trafficking ( Poteryaev et al., 2007 ).
B. Methods for Construction of Transgenic ArraysCloning
Fire et al. developed a useful tool kit which includes a comprehensive set of
commercially available expression vectors ( http://www.addgene.org/ ). These vec-
tors allow for the fusion of gene sequences with a variety of fluorescent variants and
ectopic and inducible promoters using standard cloning techniques. Alternative
cloning systems based on recombination cloning are also available and replace
traditional restriction-enzyme cloning ( Hope et al., 2004; Lamesch et al., 2004;
Luan et al., 2004; Rual et al., 2004 ). These techniques allow for modification and
assembly of multiple components or ''modules'' enabling the user to rapidly recom-
bine different parts of the transgene ( Merritt and Seydoux, 2010 ). With this method,
a library of different experimental reagents, such as tissue-specific constructs can be
assembled relatively easily.
1. Advantages and Disadvantages
Cloning is a standard laboratory practice and results in the production of a
construct that can be maintained and amplified as a renewable resource. Such clones
can be used in additional engineering steps to quickly develop a suit on constructs for
in-depth analysis. A drawback to this approach is the technical limitations on insert
size precluding the use for large genes or genomic elements.
2. PCR Stitching
A simple and effective method for engineering reporter constructs is to use the
PCR ''stitching'' method. PCR stitching is a method for joining two or more separate
DNA sequences in a single PCR reaction (for an in-depth description of the tech-
nique see Boulin et al., 2006 ). This technique relies on a short region of homology
between the separate sequences acting on internal primer site ( Fig. 10 ). The resulting
fused sequence can be purified or often injected directly into animals. Since its
development, this technique has been used extensively in C. elegans for both small
and large-scale expression analysis ( Dupuy et al., 2007; Gallo et al., 2006; Huang
et al., 2007; Zhao et al., 2004a, 2004b ).
3. Advantages and Disadvantages
PCR stitching is a straightforward approach that does not require any specialized
reagents or techniques. Constructs can be rapidly generated and are easily modified
to suit specific purposes. There are however limitations on the size of fusion
sequences that can be easily produced and this approach results in a finite amount