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(c) Low-copy expression of transgenes by microparticle bombardment: This
method has been widely used to generate integrated transgenes, especially
for gene expression in early embryos and the germ line. With this technique,
DNA is bound to gold particles and shot into worms using a biolistic bom-
bardment instrument or gene gun (Biorad). The integrated transgenes usually
contain only a few copies of the DNA of interest ( Praitis et al., 2001 ).
(d) Single-copy expression of transgenes by Mos-mediated integration: MosSci
(Mos1 mediated single-copy transgene insertion) was developed based on the
demonstration that the Drosophila Mos transposon is active in C. elegans
( Bessereau et al.,2001 ).Mos1encodes a transposase that is not present in the
genetic background of C. elegans. Transposition is induced by coexpressing
Mos1 transposase and the Mos1 transposon. MosSci has become widely used
for making single-copy integration of transgene. The insertion sites of the
transgene are fixed by the Mos sites and can be found in Fig. 2 and
Supplemental methods of Frokjaer-Jensen et al. (2008) . A slight limitation
of single-copy insertion is that the expression level of the reporter is not
sufficiently high to be observed in visual inspection. Conceivably, manipu-
lating the strength of the promoters or copy numbers of XFP tag could increase
the expression levels for such single-copy insertion of transgene reporters.
B. Labeling Neuronal Proteins and Subcellular Structures
Cytosolic free GFP labeling reveals the overall morphology of the neuron, and
allows assessment of neuronal fate and differentiation. Because neurons are polar-
ized cells, an area of intense study in neuronal development is how polarity of
neurons is determined, and how a compartment is established. Most proteins can
be tagged with XFP while maintaining their physiological functions. Additional
modifications can make tagged proteins to be further useful for revealing the
dynamics of subcellular compartments. The ease of observing tagged proteins in
liveworms has made C. elegans a prime model organism to investigate the molecular
pathways regulating the subcellular structure formation.
(1) Labeling proteins: fluorescent proteins are usually added to the N- or C-termi-
nus, or occasionally the middle of the protein of interest. The principle for
selecting the tagging position is that fusion proteins should retain similar func-
tions to endogenous proteins. Besides regular XFP as tags, photoactivatable or
photoconvertible fluorescent proteins can also be used as tags to observe dynam-
ics of proteins (such as: transport, translation, and degradation) ( Matsuda et al.,
2008; Patterson and Lippincott-Schwartz, 2002; Yampolsky et al., 2008 ).
(2) Labeling subcellular structures: Subcellular structures such as the Golgi, the
nucleus, and synapses have unique protein and lipid constituents. For example,
the hallmark of synapses is that synaptic vesicles are clustered near active zones
at presynaptic terminals, and receptors are concentrated at the postsynaptic
region ( Jin, 2005; Zhai and Bellen, 2004 ). The initial success of labeling
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