The promoter 2 reporter gene construct consisted of 2.3 kb upstream of the predicted gei-8a start codon and included exon 1 and 64 bp of exon 2. The expression of this reporter gene was observed in all larval stages starting at the L1 stage Seliciclib FDA and continuing through adulthood where expression was primarily observed in neurons of the pharyngeal nerve ring, head neurons, tail neurons and the egg-laying muscles. The promoter 3 reporter gene construct contained 6.2 kb upstream of the predicted gei-8a start codon, covering both promoter regions 1, 2 and exons 1, 2 and a part of exon 3; GFP sequences were derived from pPD95.75 by SOEing [28] and did not contain a nuclear localization signal. Expression of this reporter gene started at the embryonic comma stage.

Larval expression was detected in pharyngeal neurons, ventral and dorsal nerve cords, tail neurons, egg-laying neurons, and egg-laying muscles. In males, GFP was observed in male-specific tail ganglia and rays. Typical examples of GEI-8::GFP cell- and tissue-specific expression are shown in Figure 4. Taken altogether, our reporter gene expression results defined multiple and distinct cis-acting regulatory regions of gei-8 that drive similar expression patterns that are present throughout development and predominantly in neurons. Expression in the germline would not be revealed by this strategy because transgenes are usually silenced in the germline [29]. However, we noted that gei-8 expression in the germline has been detected by Y. Kohara��s in situ hybridization results accessible in the Kohara in situ database NEXTDB (http://nematode.

lab.nig.ac.jp). Figure 4 Analysis of gei-8 expression using transgenic lines. Loss of gei-8 Results in Mutant Phenotypes We obtained the VC1213 strain harboring a gei-8(ok1671) deletion allele generated by the C. elegans Knockout Consortium. The mutation was initially characterized as a 1095 bp deletion/45 bp insertion affecting exons 7 and 8 of gei-8a, removing the intron between them. We verified the size and location of the deletion by PCR genomic amplification from mutant animals and showed that the inserted sequences are identical to a 45 bp region from exon 7 starting at position 1550 of the predicted gei-8a isoform cDNA sequence.

Sequencing the gei-8(ok1671) cDNA revealed a stop codon present in the gei-8(ok1671) transcript at position 663, giving rise to a predicted protein containing SANT1 and SANT2 domains, but missing the majority of the putative NR interaction sites at the C-terminus of the protein. The mutant mRNA was detected in homozygous gei-8(ok1671) GSK-3 animals using RT-PCR at levels similar to wild-type animals, suggesting the premature stop codon may be bypassed in some transcripts by alternative splicing or that the premature stop codon is not efficiently recognized by nonsense mediated decay [30]. Thus, truncated GEI-8 protein may be present in homozygous mutant larvae.