Biology Reference
In-Depth Information
the transparency of the animal at all life stages, the reporter of choice is GFP or other
fluorescent proteins such as the GFP variants YFP and CFP ( Miller et al., 1999 ), or
the red fluorescent proteins dsRed and its faster-folding, monomeric variant,
mCherry ( Shaner et al., 2004 ).
The most important consideration in constructing a transcriptional reporter is the
amount of predicted regulatory sequence to include, as this will be the primary
determinant of transcriptional regulation of most genes. Because the genome
sequence of C. elegans is completely known, researchers can examine the physical
map around an uncharacterized gene on WormBase and select as large a region as is
practical, typically some 3-10 kbp or to the next upstream gene ( Dupuy et al., 2004b;
Mounsey et al., 1999 ). Comparison of noncoding sequences in orthologous genes
across sequenced genomes has also been helpful in identifying key regulatory
elements ( Elemento and Tavazoie, 2005; Kuntz et al., 2008 ). If it is found that the
nearest upstream gene is a very short distance (100-400 bp) from the start of the gene
of interest, it is possible that the two genes are in an operon ( Zorio et al., 1994 ), in
which case the promoter sequences will lie upstream of the most 5 0 transcript.
Regulatory sequences can also be found in introns, so transcriptional fusions may
need to include these sequences ( Okkema et al., 1993 ). Common methods of con-
structing reporters will be described later.
To aid in determining the timing and tissue- or cell-specificity of gene expression,
it is useful to include sequences that direct the transgene product to the nucleus. Both
the SV40 nuclear-localization signal (NLS) and the coding sequence for a histone
have been used to concentrate signals in nuclei to facilitate cell identification ( Fire
et al., 1990; Strome et al., 2001 ). The histone tags have the advantage that they stay
with chromosomes during mitosis. When combined with other transgenic lines for
which expression patterns are well characterized, a precise cell-expression pattern
can be determined. The advent of new software combined with four-dimensional
imaging using confocal microscopy has made this type of analysis technically
simpler and more sophisticated ( Murray et al., 2008 ).
B. Analysis of Protein Localization
Where the subcellular localization of a protein is being studied, the transgene can
be engineered to carry most (or all) of the coding region for the gene of interest,
tagged to a reporter construct ( Fig. 2 ). To be assured that function of the protein is not
affected by the reporter ( Prasher et al., 1992 ), it is wise to design constructs where the
tag is inserted in different positions in the coding sequence. To ensure that the
construct is functioning like the endogenous protein, the transgene should be assayed
for its ability to rescue the mutant phenotype, if a mutant is available, or for the
anticipated behavior following ectopic overexpression.
Finally, because of the possibility that the 3 0 UTR of a gene might be under control
of a micro-RNA (miRNA), inclusion of the gene ' snative3 0 UTR may be required for
the construct to reflect the expression of endogenous protein. Predictions of miRNA
Search WWH ::




Custom Search