Affinity Purification of Protein Complexes in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 293

and TH315 ( Fig. 3D ). However, incomplete rescue of the Unc phenotype makes the
identification of transformants more challenging. The target gene can be inserted in
these vectors at the 3 0 end of GFP by either Gateway cloning (TH315) or conven-
tional cloning (TH314). To fuse the protein of interest at the C-terminus to the LAP
tag either pAZ-GFPc (truncated unc-119) or pEZ-13 (full length unc-119) can be
used ( Fig. 3D ). The LAP tag can easily be transferred into other vectors using the
cassette present in pIC26.
Instead of fusing both tags to one protein, it is also possible to fuse them to different
members of the same protein complex. Such a ''split'' TAP tag was used to isolate
new binding partners of the integral membrane nicotinic acetylcholine receptor
(nAChR) ( Gottschalk et al., 2005 ). Additional epitopes have been implemented for
tandem affinity purifications ( Polanowska et al., 2004; Schaffer et al., 2010 ).
D. Introduction of Transgenes for Expression of Tagged Proteins
For somatic expression of transgenes, heritable and repetitive extrachromosomal
arrays are often sufficient; for example, Gottschalk et al. used an array to express
TAP-tagged nAChR subunits ( Gottschalk et al., 2005 ). Injecting DNA in the
C. elegans germline will generate extrachromosomal arrays ( Mello and Fire,
1995; Mello et al., 1991 ). However, a transgene in an array is typically overexpressed
in somatic cells and rapidly silenced in germ cells ( Kelly and Fire, 1998; Seydoux
and Strome, 1999 ). The variable degree of heritability of the arrays can also make it
difficult to obtain sufficient material from large-scale cultures.
An alternative to arrays is ballistic bombardment where small transgene-coated
gold particles are introduced into the worm tissue at high speed ( Praitis et al., 2001 ).
Bombardments are performed in the DP38 strain that carries a mutation in the unc-
119 gene. The DP38 strain is unable to move and does not transition to the dauer
stage. A copy of the unc-119(+) gene is introduced in the same vector as the
transgene and transformants are identified by wild-type movement and dauer for-
mation. Ballistic bombardment yields low-copy number integrations at random sites
in the genome. Bombarded transgenes may not be expressed at the endogenous level
nor at all relevant developmental stages. Another drawback of generating transgenic
lines by ballistic bombardment is that the integration sites are different for each
transgene making it difficult to compare wild-type and engineered mutants. Detailed
procedure for ballistic bombardment is described in Green et al. (2008) and on the
Seydoux laboratory website ( http://www.bs.jhmi.edu/MBG/SeydouxLab/vectors/
index.html ) .
A recent technique, MosSCI (Mos1 mediated Single Copy transgene Insertion)
circumvents the problems associated with arrays and bombarded lines by directing
the transgene at a fixed locus in the genome ( Frokjaer-Jensen et al., 2008 ).
Transformants are identified using the same strategy as for ballistic bombardment:
the injected strain contains an unc-119(ed3) mutation that is rescued by introducing
the wild-type unc-119(+) gene on the vector harboring the transgene. Description of