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specificity of the promoter, type of fluorescent protein reporters, choice of 3
0
UTR,
forms of DNA for transformation (circular plasmids or linear DNA fragments), and
method of generating transgenic worms. The following is a general guideline for
making transgenic worms to label neurons by XFP.
(1) Reporter construction:
(a) Choose a neuronal promoter: To label a specific neuron, one needs to find a
promoter that can be strongly activated in the target neuron, but not in
others. In C. elegans, most promoters refer to a few hundred base pairs to
3-5 kilobases of sequences 5
0
to the starting ATG. Extensive information
regarding tissue and cell specificity of many promoters is provided in
wormbase and C. elegans modENCODE (
Celniker et al., 2009; Gerstein
et al., 2010
). For neuron-type specific promoters, a good place to start is the
supplemental table in
Chelur and Chalfie (2007)
.
(b) Choose a reporter: Almost all fluorescent proteins are suitable for labeling
neurons. One usually begins with GFP, as it expresses well in most cells and
rarely forms aggregates. Most XFP reporters are usually stable. For analyz-
ing dynamic regulation, several modifications help to destabilize fluores-
cent proteins. For example, the PEST domain is a target of the ubiquitin
proteasome system (UPS), and experiments showed that adding the PEST
domain to the C-terminus of GFP greatly reduces the half life of GFP (
Frand
et al., 2005; Li et al., 1998
). Similarly, tagging GFP with a ubiquitin E3
ligase Ring finger domain can destabilize GFP (
Bounoutas et al., 2010;
Poyurovsky et al., 2003
)
(c) Choose a 3
0
UTR: The 3
0
UTR plays important regulatory roles in gene
expression. For stable and high-levels of expression, one of the most used
3
0
UTRs is from the unc-54 myosin gene. Increasing evidence shows that
important regulatory information may also be conveyed by the gene-spe-
cific 3
0
UTR. In general, one relies on cDNA clones to identify 3
0
UTRs. A
recent genomic study has revealed extensive 3
0
UTR variants for a given
gene (
Mangone et al., 2010
), and a search in wormbase is always helpful
when deciding which 3
0
UTR to include in a reporter design.
(2) Making transgenes:
(a) Forms of DNA: Both circular plasmids and linear DNA fragments (such as
amplified by PCR) work well in generating transgenic worms. In traditional
germ line transformation procedure, the transgene expression levels can be
controlled by the concentration of DNA (
Mello et al.,1991
). Recent studies
have suggested that using vector-free DNA fragments may improve transgene
expression levels and tissue specificities (
Etchberger and Hobert, 2008
).
(b) High-copy expression of transgenes by germ line transformation: The most
common method to generate transgenic worms is microinjecting DNA of
interest into the gonads of young adult hermaphrodites (
Mello et al., 1991
).
DNA usually forms extrachomosomal arrays that are stably transmitted, but
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