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a single cell. Multiple methods have recently been developed for labeling individual
neurons using two or more promoters with overlapping cell-type expression.
(1) The Cre-loxP recombination system: Cre-LoxP recombination refers to recom-
bining a specific sequence of DNA with the help of an enzyme called Cre
recombinase, which is widely used in mammalian systems to generate knockout
or transgenic animals. Recently, the Cre-loxP recombination method has also
been used successfully in C. elegans to label a single neuron called RMG, which
has no specific promoter available ( Macosko et al., 2009 ). The general strategy is
shown in Fig. 3 B; the key is to find two promoters that exhibit overlapping
activities in the neurons of interest. Two transgenic worms are generated: One
expresses Cre under promoter A (Promoter A::Cre) and the other expresses lox-
STOP codon-lox:: GFP under promoter B (Promoter B::Lox-Stop-Lox::GFP). In
the Promoter B::Lox-STOP-Lox::GFP transgene, one stop codon, three poly-A
sequence and two mRNA cleavage sequences are inserted between the two Lox
sites to ensure that GFP can not be expressed in the single-transgenic worms. In
the double transgenic worms (Promoter A::Cre; Promoter B::Lox-STOP-Lox::
GFP), Cre can mediate recombination between the two Lox sites to eliminate the
transcriptional stop sequence, and allow GFP expression ( Fig. 3 B).
(2) Reconstituted GFP: Another way to label specific neurons utilizes the prop-
erties of reconstituted fluorescent proteins ( Zhang et al.,2004 ). An early
version of split GFP uses two fragments (GFP(1-157) and GFP(158-238))
which can restore fluorescence when they are reconstituted by the leucine
zipper domain dimerization ( Hu et al., 2002 ). As shown in Fig. 3 C, the N
terminus GFP fragment(1-157) is fused to a leucine zipper domain, modified
from the leucine zipper domains of mammalian Fos and Jun proteins, in C
terminal, and the C terminus GFP fragment (158-238) is fused to a leucine
zipper (LZ) domain in N terminal ( Zhang et al., 2004 ). Neither N-GFP(1-
157)-LZ nor LZ-C-GFP(158-238) alone can produce fluorescence, but when
N-GFP-LZ and LZ-C-GFP are coexpressed in one cell, GFP can be recon-
stituted through the dimerization of LZ domains ( Fig. 3 C) ( Zhang et al.,
2004 ). Using this reconstituted fluorescent protein method, specific types
of neurons can be labeled by two different promoters driving N-GFP-LZ
and LZ-C-GFP ( Zhang et al., 2004 ). This method is also referred as
''bimolecular fluorescence complementation (BiFC),'' which allows for
detecting protein interactions in vivo by fusing N-GFP(1-157) and
C-GFP(158-238) to different proteins ( Hu et al., 2002, 2005 ).
Recently, a variation of this split-GFP based combinatorial labeling has
been developed for visualizing synapses between specific pair of neurons, a
method named GRASP for GFP Reconstitution across Synaptic Partners
( Feinberg et al., 2008 ). The GRASP method utilizes the superfolder split-
GFP, which has a different split module: GFP-A (aa 1-214) and GFP-B
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