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(aa 215-230) (
Pedelacq et al., 2006
). Fragment A is fused to the extracellular
juxtamembrane position of a surface protein, and expressed in postsynaptic
cells. Fragment B is inserted after an artificial signal peptide followed by the
coding sequence of a presynaptic terminal member protein PTP-3A, and
expressed in the presynaptic cells (
Feinberg et al., 2008
). If the two mem-
brane proteins are localized to close proximity, such as synaptic junctions,
the split GFP fragments A and B are close enough to generate fluorescence,
marking the synaptic region (
Fig. 3
D). The GRASP method can also be
adapted to label any types of cellular junctions.
D. Questions and Answers
- How does one specifically label neurons of interest?
If there is not a specific promoter to label neurons of interest, systematic promoter
bashing is necessary to define regulatory sequences for labeling specific neurons.
- How can one be sure about labeling specificity?
Ectopic or improper expression is an inherent problem in transgene formation.
A caution is that one should always confirm the cell-type-specificity of a
reporter by analyzing multiple lines. In some cases, if the tagging of protein
of interest does not allow verification of cell type, one may consider the
strategy to include an operon-reporter, SL2-XFP,
in the same construct
(
Macosko et al., 2009
).
- How does one get stable and good expression of transgenes?
Some extrachromosomal transgenes can be silenced in succession of passage. The
cause is largely unknown, though repetitive elements are suspected to be a likely
trigger. To overcome this problem, integrated transgenes (multiple copies or
single copy) are a good option. One additional suggestion is to include complex
DNA, such as random genomic DNA, in making transgenes.
- How does one minimize transgenic labeling artifacts?
One common concern for using transgenes is that transgenic expression could
alter the normal development or function of a cell. To minimize such effects, it is
advised to perform functional assays (e.g. rescue the null allele phenotypes) to
confirm that the transgenes are functional in a manner similar to the wild-type
proteins. One should also test multiple transgenic lines generated from different
DNA concentrations. Another possible artifact for using fluorescent protein
reporters is from the extremly stable fluorescent proteins. For example gene A
is only expressed in the nervous system at early larval stages, but not in adult.
However, when using standard GFP as a reporter to observe the expression pattern
of A gene, you may see that the reporter of gene A is expressed in adult stages due
to the persistent expression of GFP. Use of an unstable GFP as a reporter is an
option in this case.
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