Whereas FMRP was highly expressed in the unaffected control line, FMRP protein expression was not detectable in any of the FXS patient fibroblasts. Interestingly, the mosaic GM05131 fibroblasts that demonstrated only partial methylation of the FMR1 promoter had undetectable protein, even though they had elevated transcript levels; it has been reported before that production of FMRP is greatly reduced in the premutation state, which may be due in part to a relative block in translation SB203580 cost caused by the presence of the 59UTR extended CGG repeat . FXS patient and control fibroblasts were reprogrammed to pluripotency using established methods . We further analyzed two iPSC 157716-52-4 clones from GM05848 , two clones from GM05131 , one clone from GM05185 and control iPSC lines from GM08330 and BJ1 . All iPSC clones had typical characteristics of human pluripotent stem cells indicating successful reprogramming , including: a) human embryonic stem cell colony-like morphology, b) alkaline phosphatase expression and immunoreactivity for OCT4 , NANOG and Stage-specific embryonic antigen-4 , c) expression of endogenous OCT4, NANOG, and REX1 , d) de-methylation of the endogenous OCT4 promoter , and e) normal karyotypes . In addition, both FXS and control iPSC clones differentiated into all three germ layers in vitro , including early neural tissue. Importantly, in concordance with the assessment of a loss of GFP expression from the retroviral vectors, analysis of transgene expression in the control and Fragile X syndrome iPSC clones using RT-PCR and primers specific for transgene cMYC, OCT4/POU5F1, KLF4, SOX2 indicated a silencing of their expression . Observation of the growth rate and the ability to remain undifferentiated in culture over many passages did not reveal any obvious qualitative differences between the unaffected control and FXS iPSC lines. We found that the two iPSC clones we generated from the GM05131 cell line appear to be derived from the two different fibroblast subpopulations. One iPSC clone had approximately 700 and the other 140 CGG repeats . These CGG-repeat lengths are similar to those detected in the heterogeneous input fibroblasts . Characterization of methylation of the FMR1 promoter region showed that, as expected, the iPSC clone 131- iPS1 had a mean CpG methylation of approximately 90%, while clone 131-iPS3 was essentially unmethylated . FMR1 expression analysis showed that there were no detectable transcripts from the fully CpG methylated 131-iPS1 clone, while the premutation 131-iPS3 clone showed increased expression compared to the unaffected controls .