Genome Engineering by Transgene- Instructed Gene Conversion in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 84

V. Discussion: MosTIC Applications and Derivatives
A. Gene Function Analysis Using MosTIC-Engineered Alleles
In our laboratory, we routinely use MosTIC to tag genes of interest and our first
study using MosTIC-engineered alleles was published recently ( Gendrel et al.,
2009 ). We characterized a protein complex involved in the clustering of acetylcho-
line receptors at the C. elegans neuromuscular junction. In this study, KI alleles were
used for genetic, cell biology, and biochemistry experiments. It bypassed the expen-
sive and time-consuming need of developing and testing antibodies specifically
recognizing each protein of interest.
In the course of our experiments, we often observe that the use of MosTIC-
engineered alleles significantly minimizes experimental artifacts due to overexpres-
sion of engineered proteins expressed from repetitive transgenes. For instance, we
expressed a functional YFP-tagged version of UNC-63, one of the subunits of an
acetylcholine receptor present at the neuromuscular junction, from repetitive tran-
genes and by MosTIC ( Gendrel et al., 2009 ). As illustrated In Fig. 6 , endogenous
Fig. 6 Expression of tagged proteins in C. elegans: repetitive transgenes versus engineered endogenous loci. (A) The
levamisole-sensitive acetylcholine receptor is detected by immunostaining of its subunit UNC-29. It is localized in the muscle
membrane at the nerve ring (nr), the dorsal nerve cord (dc) and the ventral nerve cord (vc). (B-E) Localization of an YFP-tagged
version of UNC-63, another subunit of the levamisole-sensitive acetylcholine receptor. (B) The tagged UNC-63 subunit is
expressed from a repetitive transgene. In the presence of this transgene, a loss-of-function allele of unc-63 is rescued indicating
that functional receptors are made and that at least some of them are located at the muscle membrane. However, most of the
tagged subunit is diffusely localized in body wall muscles (bwm) (af: gut autofluorescence). (C) Confocal imaging of a strain
homozygous for a MosTIC-engineered allele expressing the same UNC-63-tagged version as in (B). Fluorescence is mostly
detected in the nerve ring and ventral and dorsal nerve cords. (D) Immunostaining characterization of the strain observed in (C).
An antibody against the YFP detects puncta in the ventral nerve cord that co-localize with the other subunits of the levamisole-
sensitive acetylcholine receptor. (Images are courtesy of C. Gally and G. Rapti). (See color plate.)