s have been reported from limited studies of patient cells. We were therefore interested to assess these parameters in our mutant iPS cells. First we compared pseudouridine content by labeling cells 
with 32P-labeled phosphate, purifying 28S rRNA and analyzing the nucleotide content by thin layer chromatography. No difference in the relative amounts of uridine and pseudouridine is seen between mutant and wild type cells. Next, we used a very sensitive pulse chase experiment in which PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19667322 cells were labelled with PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19666584 L- methionine and chased with cold methionine. Since methionine pools are low the experiment is very sensitive in detecting species with increased half lives and has been used to detect rRNA processing defects in animal models of X-linked DC. A353V and L37 cells did not show any differences in the kinetics of rRNA processing. Then, we checked the expression of some snoRNAs in these iPS cells, including some H/ACA, scaRNA and C/D snoRNA. We found that most of the H/ACA snoRNAs and scaRNAs we investigated were decreased in mutant cells while all C/D snoRNA were unchanged compared to WT iPS cells. In addition mobility shift under partially denaturing conditions has been shown to be a sensitive indicator of differences in pseudouridylation. While rRNAs from WT and mutant iPS cells both showed the denaturation dependent shift there was no mobility difference between them. Variable rescue of Peretinoin web impaired telomerase function by expressing WT dyskerin We were interested in rescuing the short telomere phenotype for several reasons. First we wanted to know if telomere shortening in DC is reversible. Second we were interested in whether DKC1 mutants would exert a dominant negative effect on the WT protein. This is not 8 / 20 Dyskeratosis Congenita iPS Cells 9 / 20 Dyskeratosis Congenita iPS Cells Fig 2. DKC1 mutant iPS cells show no significant defects in ribosome biogenesis. A: measurement of pseudouridine in 28S rRNA from WT and mutant iPS cells. iPS cells were labeled with 32P-labeled orthophosphate and 28S rRNA was gel-purified. After digestion with RNase T2, each sample was separated by two-dimensional TLC. The positions of the labeled ribonucleotides are indicated. Ap: Adenine, Cp: Cytosine, Gp: Guanine, Up: Uridine, p: Pseudouridine: B: Pulsechase labeling experiments of rRNA isolated from WT, A353V and L37 iPS cells. Cells were labeled with L- methionine for 30 min and then chased in nonradioactive medium for the times shown. The RNA was separated on a 1.25% agarose gel, transferred to a nylon filter, and exposed to x-ray film. C: Real-time RT/PCR results of some H/ACA snoRNAs of WT and DKC1 mutant iPS cells. Results were expressed relative to GAPDH RNA. The combined results of 3 independent experiments are shown, the error bars show standard deviation. doi:10.1371/journal.pone.0127414.g002 known because in either male or female cells only one allele is expressed per cell. Finally if we could lengthen telomeres by inserting WT dyskerin and then excise the WT dyskerin cassette we would have a cell with a DKC1 mutation and long telomeres, in which the early stages of telomere shortening due to DKC1 mutations could be investigated. We used a zinc finger nuclease recombination system to introduce a Flag-tagged WT DKC1 cDNA expression vector into the “safe harbor” AAVS1 locus of A353V and L37 cells. We obtained some clones with a single heterozygous integration according to Southern blot analysis and studied two corrected lines for both mutations
