We necessary injected fusion PCR products, without purification, into the gonad of young adult hermaphrodites of CB00907 at a concentration of 10 ng uL together with 100 ng uL dpy 5 plasmid in 1XTE buffer to gener ate extrachromosomal arrays. On average, 25 30 P0 dpy 5 hermaphrodites were injected with each pSAC,GFP construct. Rescued Dpy 5 mutant pheno type was indicative of transformants. These wild type looking F1 progeny were plated individually and screened for the presence of wild type F2 progeny. On average, we obtained three to five lines yielding at least 30% wild type progeny. Aware of the mosaicism issues associated with extrachromosomal concatamer arrays, we analyzed at least 30 replicates for each developmen tal stage.

Once these lines were genotyped and con firmed to have similar expression patterns, one line for each construct was frozen and kept as a transformed stock. Genotyping was performed using promoter spe cific primer and GFP specific primer. In vivo analysis and imaging of pSAC,GFP transgenic lines For each transgenic line, we prepared mixed staged population of worms and immobilized them in 100 mM sodium azide immediately before imaging. Initially, worms were analyzed using a Zeiss Axioskop equipped with QImaging camera to confirm the consis tency of expression patterns between the transgenic lines. Then more detailed analysis, which involved tak ing stacks of confocal images with 0. 2 0. 5 um between focal planes, was performed using Quorum WaveFX Spinning Disk system mounted on a Zeiss Axioplan microscope.

All images were taken at 400X, image acquisition and analysis was performed using a Volocity software Anacetrapib package. Viability measurement For all the double and single mutants, five L4 wild type looking worms were individually plated at 20 C. The worms were transferred to fresh plates every 12 hours and the plates were scored. Total numbers of eggs laid defined the brood sizes. The eggs that did not hatch in 24 hours were scored as embryonic arrest. The eggs that hatched but did not reach adulthood were scored as lar val arrest. The progeny that developed to adulthood were scored for incidence of males. The percent fertility was determined by individually plating all progeny that developed to adulthood. All of the single and double mutants were then analyzed in a SCM,GFP background for number of seam cells by using Zeiss Axioskop equipped with QImaging. Human embryonic stem cell lines were derived from the inner cell mass of human blastocysts, and were cultured on mouse embryonic fibroblast feeder to maintain undifferentiated growth, extensive renewal capacity, and pluripotency, including the ability to form teratomas in SCID mice and embryoid bodies in vitro.