Transgenesis in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 166

experiments. A discussion of markers commonly used for making transgenic lines is
included below.
In one variant of complementation of a chromosomal mutation, the transgene is
used to rescue an animal with a phenotype resulting from treatment with RNAi. In
this case, the transgene is engineered to have resistance to the RNAi effect. When the
transgene is combined with tissue-specific or altered promoters, this strategy allows
for the assessment of genes that act in multiple tissues or at different stages of
development ( Green et al., 2008 ).
Introduction of transgenic sequences is also useful for functional characterization
of a gene ( Fig. 2 ). For example, a transgenic construct carrying an altered version of a
gene can be assayed to determine if it rescues some or all functions provided by the
wild-type gene product (i.e., a sufficiency assay). Introduction of predicted ortho-
logs or paralogs, under the control of a C. elegans promoter, can determine conser-
vation of functional domains or gene products. Transgenic constructs containing
altered or entirely different regulatory sequences can be used to examine the con-
sequences of ectopic or reduced expression. An altered transgene can be designed to
test the function of a particular splice isoform. The transgenes can also be fused to
sequences that target them to specific subcellular locations or cause them to be
secreted. Several sequences are known that provide subcellular targeting, which
includes nuclear localization (SV40 NLS or histone), membrane targeting, secre-
tion, or mitochondrial import ( Fire et al., 1990; Portereiko and Mango, 2001; Strome
et al., 2001 and Fire Lab Vector information; Addgene). Each of these strategies
permits the researcher to manipulate gene activity in order to better characterize the
function of a gene of interest.
D. Marking Tissues and Cells for Other Manipulations
The rich variety of existing transgenes allows investigators to mark tissues so that
they are more easily followed for live cell imaging, to characterize the effect of
environmental or genetic manipulation on development of particular cell types or
substructures, or to follow cells in a non-natural context, such as after dissociating
embryonic blastomeres. A large number of well-characterized promoters can be
searched indirectly by expression in particular tissues, stages, or cells on
WormBase. The use of reporters in combinations allows the detection of multiple
expression patterns in the same animal, an analysis that is particularly useful for
determining lineage-specific expression. Reporters of differing absorption/emission
spectra can be used, such as the combination of CFP and YFP, or mCherry with GFP.
With mCherry and GFP using standard TRITC and FITC filter sets, the two reporter
signals show very little overlap. With CFP and YFP, specific filter sets are used to
prevent cross-detection ( Miller et al., 1999 ). Signals in strains expressing all four
fluorescent proteins can be discerned because of the behavior of each fluorescent
protein in each optimal filter set ( Table I I ). This analysis permits both the deeper
understanding of mutant phenotypes and the expression patterns of newly charac-
terized gene products.