Figure 3

Recombineering-mediated construction of a fosmid-based ceh-12 :: gfp translational reporter gene fusion in MW005. Panel A. A 33.5 kb stretch of C. elegans chromosome I, from bps 5,815,150 to 5,848,663, equivalent to the insert of genomic clone WRM0611dH08, illustrating the number and orientation of genes predicted within this region (ceh-12 boxed in red). Panel B. Expanded view of ceh-12 illustrating the internal exon- (magenta box) intron (line) organization (scale bar = 100 bp). Panel C. Cartoon (not to scale) representing the pCC1FOS-based C. elegans genomic target clone WRM0611dH08 and the final recombineered construct fUL#SB28. A gfp reporter sequence containing four exons (green boxes) was seamlessly inserted, by counter-selection recombineering in MW005, at the 3' end of the ceh-12 gene contained within WRM0611dH08 to give fosmid clone fUL#SB28 containing ceh-12::gfp. Panel D. Expression of ceh-12::gfp is restricted to VB motorneurons in the ventral nerve cord (arrowhead, left panel fluorescence image). Arrows indicate the vulva on the ventral side of the worm. (Micrographs were captured with Chroma Technology Corp. filter set 41017 on a Leica DMR microscope equipped with DIC optics, a Hamamatsu ORCA ER digital camera and Improvision Openlab software at 400× magnification with a 2 sec exposure). The right panel shows a DIC brightfield image of the same specimen.