br Resource details Human skin fibroblasts isolated from a y

Resource details Human skin fibroblasts isolated from a 26year old Puerto Rican female HPS1 patient were obtained from the Coriell Cell Repositories (Camden, NJ). The parental line, GM14609, carries a homozygous 16bp duplication (codons 491–496) in exon 15 of the HPS1 gene. The HG-9-91-01 were transduced with STEMCCA Cre-excisable constitutive polycistronic lentivirus expressing Oct4, Klf4, Sox2, and c-myc. Cre-mediated excision of the vector following reprogramming was demonstrated (Fig. 1A). The mutation was validated by DNA sequencing and quantitative PCR (qPCR) (Fig. 1B). The cells exhibited a normal karyotype (46, XX) upon G-band analysis (Fig. 1C). Pluripotency was verified by gene expression of stem cell markers DMNT3B, REX1, ABCG2, OCT4, NANOG, and SOX2 using qPCR (Fig. 1E). In addition, expression of pluripotent surface markers at single cell resolution was confirmed by flow cytometry (Fig. 1D). Differentiation capacity into three germ layers was confirmed by in vivo teratoma formation (Fig. 1F).
Materials and methods
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We generated KCL024 clinical grade hESC line following protocols, established previously (Ilic et al., 2012; Stephenson et al., 2012). The expression of the pluripotency markers was tested after freeze/thaw cycle. Differentiation potential into three germ layers was verified in vitro.
Materials and methods
Author disclosure statement
Acknowledgments This work was supported by the UK Medical Research Council grants G0701172 and G0801061. We thank Dr. Yacoub Khalaf, Director of the Assisted Conception Unit of Guy\'s and St. Thomas\' NHS Foundation Trust and his staff for supporting the research program. We are especially indebted to Prof. Peter Braude and to the patients who donated embryos.
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Resource details To generate BJNhem20-OCIAD1-Tet-ON cell line, we transfected BJNhem20 with pCAG-Tet-On (pCAG-rtTAOn) and pTRE-Tight-OCIAD1 by microporation at 1100V, 30ms pulse width and 1 pulse number (see Fig. 1A). A stable hESC line was generated after subjecting these transfected cells to hygromycin and geneticin selection for ten days. The pTRE-Tight-OCIAD1 construct was generated by cloning OCIAD1 Open Reading Frame amplicon from amino acids 1 to 245 between EcoRI and NotI sites using appropriate primers. Induction of OCIAD1 expression was done using doxycyline (300ng/ml).
Materials and methods
Acknowledgments This project was funded by the Department of Biotechnology, Government of India (Grant no. BT/PR11115/MED/31/40/2008) and the Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore. We thank Ridim D. Mote for generating pTRE-Tight-OCIAD1 plasmid construct.
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We generated KCL026 clinical grade hESC line following protocols, established previously (Ilic et al., 2012; Stephenson et al., 2012). The expression of the pluripotency markers was tested after freeze/thaw cycle. Differentiation potential into three germ layers was verified in vitro.
Materials and methods
Author disclosure statement
Acknowledgments This work was supported by the UK Medical Research Council grants G0701172 and G0801061. We thank Dr. Yacoub Khalaf, Director of the Assisted Conception Unit of Guy\'s and St Thomas\' NHS Foundation Trust and his staff for supporting the research program. We are especially indebted to Prof Peter Braude and to the patients who donated embryos.
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Materials and methods
Acknowledgments The work in the author\'s laboratory is currently supported by the grant from NIH (R01GM093335). XL conceived the study. HTL, LL, CR, FTB and XL performed the experiments. XL wrote the manuscript.
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We generated KCL025 clinical grade hESC line following protocols, established previously (Ilic et al., 2012; Stephenson et al., 2012). The expression of the pluripotency markers was tested after freeze/thaw cycle. Differentiation potential into three germ layers was verified in vitro.