Transgenesis in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 167

Table II
Cross-detection of popular fluorescent reporters in common filter sets

Appearance in filter set

TRITC (Chroma 31002) * YFP (Chroma 41029) * GFP (Omega XF100-2) * CFP (Chroma 31053) * Fluorophore

mCherry Red Faint red Not visible Not visible

dsRed Red Orange Faint orange Not visible

YFP Faint red Green Green Not visible

GFP Not visible Green Green Green

CFP Not visible Not visible Green Cyan

*

Specifications of the various filters can be found on the manufacturer ' s websites ( http://www.chroma.com ; http://www.
omegafilters.com ).

E. Disruption of Gene Activity: RNA Interference
Extrachromosomal arrays have been used to generate dsRNA in vivo that can elicit
RNAi. Because of the ability of RNAi to spread among tissues (systemic RNAi),
expression of the RNAi construct does not need to occur throughout the entire
animal. The constructs can consist of separate sense and antisense RNA transgenes,
or constructs expressing a single hairpin (stem-loop) construct ( Fig. 2 ). Expressing
hairpin constructs within neurons has been effective for RNAi knockdown of genes
that might be more difficult to achieve by feeding-based RNAi ( Johnson et al., 2005;
Tavernarakis et al., 2000 ). One difficulty with hairpin constructs is that the DNA
constructs are unstable in E. coli; this limitation may be overcome by using stem-
loops with introns in the loop portion, or the use of E. coli strains that are more
tolerant to such structures (e.g., SURE2 cells; Stratagene).

F. Mosaic Analysis of Gene Function
Researchers often need to test the requirement of a gene within the context of a
subset of its normal expression. This may be done to avoid a requirement for the
function of a gene in an earlier developmental stage, or to test if gene function is cell-
autonomous. Restricted expression of a gene product can be achieved using a variety
of techniques, including creating mosaics through loss of extrachromosomal arrays
carrying a gene of interest or by fusion of the gene of interest to a specific promoter.
A number of other strategies that promise to permit even more sophisticated analysis
of tissue-specific gene expression have also been recently developed ( Table III ).
The classic approach to making mosaic animals in C. elegans is to use extrachro-
mosomal arrays as surrogate chromosomal free duplications, which experience
mitotic loss within a single animal at a low frequency (0.1 10 3 to 5 10 3 loss
per cell division) ( Hedgecock and Herman, 1995; Lackner et al., 1994; Miller et al.,
1996; Yochem and Herman, 2005 ). Extrachomosomal arrays have an advantage over
free duplications because the researcher can determine their composition. To