Transgenesis in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 173

III. Construction of Transgenes
An excellent description of the many considerations for construction of plasmid
reporters can be found in Boulin et al. (2006); Mounsey et al. (1999) , which we have
updated here. There do not appear to be any sequence requirements for the stable
inheritance of arrays in C. elegans ( Mello et al., 1991 ), as DNA from plasmids or
phage, for example, appears to be incorporated into arrays. Hence, standard molec-
ular biology techniques can be used to construct most transgenes. When segments of
wild-type DNA are needed, polymerase chain reaction (PCR) can be used to amplify
directly from genomic DNA, or larger clones, such as a cosmids or fosmids, can be
ordered and used for either direct subcloning or PCR. Information on ordering
clones is available on WormBase ( Table I ). Researchers are cautioned that some
larger clones are unstable when propagated in bacteria or yeast, such that a particular
isolate of a clone could be missing regions of DNA. When working with these
constructs it is always advisable to check that sequences have not been lost.
For simple reporter fusions of zygotically expressed genes, it is usually sufficient
to clone a suitable upstream promoter fragment (3-10 kbp is a good start, without
taking sequences from the neighboring gene upstream) along with a small part of the
coding region. The fragment is cloned into one of the available GFP vectors (gen-
erated by the laboratory of Andrew Fire). These vectors supply a useful polylinker,
synthetic introns to increase expression, and a 3 0 UTR from the unc-54 gene. Variants
are available that encode other fluorescent proteins (YFP or CFP), include a nuclear
localization signal (NLS), or are a fusion to both GFP and lacZ. Other vectors use a
histone H2B coding sequence as a more effective means to localize GFP to nuclei. A
number of useful vectors as well as additional documentation from the Fire lab can
be obtained from Addgene ( http://www.addgene.org ) . Where an investigator
hypothesizes sequence requirements that necessitate a much larger context for the
reporter (e.g., tens of kilobasepairs), manipulations can be performed using recom-
bination in yeast or fosmids ( Dolphin and Hope, 2006; Tursun et al., 2009; Zhang
et al., 2008 ).
Other applications of transgenes, such as the fusion of a promoter to a different
downstream sequence, will require approaches unique to each application ( Fig. 2 ).
Additional resources available to the research community can simplify cloning or
allow rapid scaling-up of construct production. For example, it is now possible to use
the Gateway recombination cloning system to fuse promoters from the ''promoter-
ome'' library into a suitable reporter. For making novel fusions of promoters to
different coding regions, clones from the promoterome can be combined with clones
from the ORFeome ( Dupuy et al., 2004a; Hope et al., 2004; Reece-Hoyes et al.,
2005 ).
For expression of heterologous coding regions, it may be cost-efficient for an
investigator to order an open reading frame to be synthesized de novo. Custom gene
synthesis can now be achieved for a relatively low cost per base pair. This would also
allow engineering for efficient expression in C. elegans, such as by the introduction
of short introns, or the selection of codons that are optimized for maximal gene