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Embryonic Stem Cells/Induced Pluripotent Stem Cells
Article first published online: 20 SEP 2012
Copyright © 2012 AlphaMed Press
Volume 30, Issue 10, pages 2199–2211, October 2012
How to Cite
Wang, C.-H., Ma, N., Lin, Y.-T., Wu, C.-C., Hsiao, M., Lu, F. L., Yu, C.-C., Chen, S.-Y. and Lu, J. (2012), A shRNA Functional Screen Reveals Nme6 and Nme7 Are Crucial for Embryonic Stem Cell Renewal. STEM CELLS, 30: 2199–2211. doi: 10.1002/stem.1203
Author contribution: C.H.W.: collection and assemble the data, data analysis and interpretation, manuscript writing, and final approval of the manuscript; N.M., L.F.L., and S.Y.C.: manuscript writing and final approval of the manuscript; Y.T.L.: collection and assemble the data, data analysis and interpretation, and final approval of the manuscript; C.C.W.: collection and assemble the data and final approval of the manuscript; M.H.: provision study material and final approval of the manuscript; C.C.Y.: collection and assemble the data and data analysis and interpretation; J.L.: data analysis and interpretation, manuscript writing, and final approval of the manuscript. C.-H.W., N.M., and Y.-T. Lin contributed equally to this article.
Disclosure of potential conflicts of interest is found at the end of this article.
First published online in STEM CELLSEXPRESS August 16, 2012.
- Issue published online: 20 SEP 2012
- Article first published online: 20 SEP 2012
- Accepted manuscript online: 16 AUG 2012 03:18PM EST
- Manuscript Accepted: 3 JUL 2012
- Manuscript Received: 14 DEC 2011
- National Health Research Institutes. Grant Number: NHRI-EX100-10025SI
- National Science Council. Grant Numbers: NSC-100-2314-B-001-002 NSC-97-2321-B-001-022-MY3, NSC-100-2321-B-001-039-
- National RNAi Core facility
- National Research Program for Biopharmaceuticals (NRPB)
Additional Supporting Information may be found in the online version of this article.
|sc-11-1192_sm_SupplFigure1.pdf||245K||Fig.S1 Selection of differentiated markers for shRNA functional screen. (A) The responsive timing of the differentiated markers after culture mESCs in a LIF-free condition. The flatten-shape morphology, expression level of Oct4, ALP activity, and high nuclear/cytoplasmic ratio are the features of ESCs. These features indicate ESC pluripotency. (B) Calculation of the sensitivity of candidate markers by the z-factor of batch variation between negative and positive controls. The well-to-well variation and the plate-to-plate variation were calculated.|
|sc-11-1192_sm_SupplFigure2.pdf||51K||Fig.S2 Neither knockdown Nme6 nor Nme7 affects the viability of MEF. Cells were infected by lentivirus with Nme shRNA (MOI=1). Cell viability was measured by Alamar Blue assay. The results were normalized against shLuc.|
|sc-11-1192_sm_SupplFigure3.pdf||120K||Fig.S3 The expression levels of ESC renewal related gene, p53, Tbx3, Nanog, Dnmt1, Dnmt3A, Sox2, Lin28, Ronin, Rest, and Tcl1 were detected by QRT-PCR. The mRNA transcripts were normalized with shLuc.|
|sc-11-1192_sm_SupplFigure4.pdf||58K||Fig.S4 The ROSA-TET knock in system. Schematic presentation of GFP (A) or Nme (B) cDNA knockin in the ROSA 26 locus. Arrow heads indicate the location of the primers used to examine if the genes knock-in in the ROSA26 locus. (C) Examination the GFP and Nme cDNA knock-in in the ROSA26 locus by PCR. PCR was performed with the GFP, Nme6, Nme7v1 knock-in stable lines.|
|sc-11-1192_sm_SupplFigure5.pdf||133K||Fig.S5 Characterization of Nme6 or Nme7 early and late responsive genes. (A) Knockdown the expression of either Nme6 or Nme7 in ESC downregulates the expression levels of ESC pluripotency and renewal factors. Two independent shRNAs of Nme6 or Nme7 were each transfected into ESCs and the expression amount of Oct4, Klf4, Myc, Dnmt3B, telomerase (Tert), and ERas were measured by QRT-PCR with three replicates at day 1, day2, and day3 post-transfection. (B) Overexpression of either Nme6 or Nme7 in ESC activates the expression amounts of ESC pluripotency and renewal factors. Vector control, Nme6, Nme7v1 and Nme7v2 were transfected into ESCs and the expression amount of the genes were measured by QRT-PCR with three replicates at day 1, day2, and day3 post-transfection.|
|sc-11-1192_sm_SupplFigure6.pdf||114K||Fig.S6 Nme6 and Nme7 are essential for teratoma formation. (A) Either shNme6 or shNme7 reduces the oncogenicity of ESCs. Transfected ESCs selection with puromycin for 5 days were counted, and 5X106 of cells were subcutaneously injected into SCID mice. The teratoma weight of ESC transfected with either shLuc, shNme6, or shNme7 were measured. (B) Histology of the pluripotency of teratoma was evaluated. The presence of the three germ layers was examined by hematoxylin-eosin staining in shLuc, shNme6, and shNme7 transfected lines.|
|sc-11-1192_sm_SupplTable1.pdf||23K||Supplementery Table 1|
|sc-11-1192_sm_SupplTable2.pdf||75K||Supplementery Table 2|
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