Transgenesis in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 171

controlling gene structure, gene product stability, or gene activity using either tissue-
specific promoters or by controlling the presence of inducers or repressors of gene
expression. One technique takes advantage of the well-characterized FLP recombi-
nase system to activate a gene of interest in specific tissues or at specific times of
development. In this strategy, the regulatory element is separated from the gene of
interest by a sequence that is cleaved upon tissue-specific expression of FLP recom-
binase, thereby activating the gene ( Davis et al., 2008; Voutev and Hubbard, 2008 ).
Until recently, the available methods for delivering transgenes, which tend to be
higher copy number and contain rearrangements, limited the effectiveness of this
technique for temporal or tissue-specific knockdown (as opposed to activation) of
gene activity. A second technique that permits control of gene expression depends on
reconstituting gene activity from two gene components, each under the control of a
specific promoter. When the two elements are combined, as would be expected in a
small number of specific cells, gene activity is restored ( Chelur and Chalfie, 2007;
Zhang et al., 2008 ). This technique is limited to genes or processes that can be
reconstituted from two components. A third technique requires altering the 3 0 UTR of
a gene and taking advantage of temperature sensitivity of nonsense-mediated decay
of RNA products to promote gene stability or decay ( Drake et al., 2003 ). However,
the effects on gene-expression levels are not always absolute. A fourth technique
depends on rescuing a heat-shock deficient hsf-1(sy441) mutant in a cell-specific
manner by controlled expression of wild-type hsf-1, permitting expression of a heat-
shock inducible promoter linked to one ' s gene of interest in only those cells ( Bacaj
and Shaham, 2007 ). This technique is limited by the temperature-sensitivity of the
process and by the transient nature of the heat-shock response. A fifth method takes
advantage of the observation that MEC-8 is required to splice mec-2 intron 9, thereby
regulating the expression of mec-2 splice variants. By creating a transgene carrying
the mec-2 intron 9 sequence upstream of a gene of interest in a mec-8(u218ts) strain,
one can regulate expression of the gene using temperature shifts. This technique was
used to control expression of the RNAi gene rde-1 to create a line with temperature-
dependent RNAi ( Calixto et al., 2010 ). Potential limitations of this technique include
the need towork in a mec-8 strain, as well as the relative stability of MEC-8 and dose-
sensitivity of the splicing event. Despite some limitations, each of these techniques
offers researchers valuable tools for selective expression of their gene of interest.
Knockdown of gene function in specific tissues can also be used to examine gene
activity. The cell-autonomous requirement for RDE-1 function in RNAi can be
exploited by providing wild-type function of rde-1 in a tissue-specific manner to
an rde-1 mutant strain. Animals then treated with RNAi to a gene of interest will
experience knockdown only in cells carrying RDE-1 function ( Qadota et al., 2007 ).
While this strategy has been used effectively for some genes, the strength of the
RNAi response can be variable.
In summary, the C. elegans researcher has a large set of techniques that can be
used to understand the role of a gene in a specific cell or developmental process,
bypass requirements at specific stages, or examine the consequences of ectopic gene
expression. The specific gene studied, the hypothesis being tested, and the