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Gene silencing using RNA interference in embryonic stem cells.

Publication ,  Journal Article
Velkey, JM; Slawny, NA; Gratsch, TE; O'Shea, KS
Published in: Methods Mol Biol
2006

Pluripotent embryonic stem (ES) cells are an important model system to examine gene expression and lineage segregation during differentiation. One powerful approach to target and inhibit gene expression, RNAi, has been applied to ES cells with the goal of teasing out the cascades of gene expression/repression that shape the early embryo. In this chapter, we describe the current understanding of the mechanisms of gene silencing by small hairpin RNAs, as well as controls and caveats to using this approach in ES cells. A consideration of synthetic vs plasmid-based RNAi vectors, design of targeting constructs, transfection of ES cells, and flow sorting of targeted cells is followed by methods for the analysis of phenotype and behavior of targeted cell populations using immunohistochemistry, reverse transcriptase polymerase chain reaction, Western blotting, and scanning electron microscopy.

Duke Scholars

Published In

Methods Mol Biol

DOI

ISSN

1064-3745

Publication Date

2006

Volume

329

Start / End Page

233 / 261

Location

United States

Related Subject Headings

  • Transfection
  • RNA, Small Interfering
  • RNA Interference
  • Pluripotent Stem Cells
  • Plasmids
  • MicroRNAs
  • Mice
  • Liposomes
  • Lipids
  • Immunohistochemistry
 

Citation

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Velkey, J. M., Slawny, N. A., Gratsch, T. E., & O’Shea, K. S. (2006). Gene silencing using RNA interference in embryonic stem cells. Methods Mol Biol, 329, 233–261. https://doi.org/10.1385/1-59745-037-5:233
Velkey, J Matthew, Nicole A. Slawny, Theresa E. Gratsch, and K Sue O’Shea. “Gene silencing using RNA interference in embryonic stem cells.Methods Mol Biol 329 (2006): 233–61. https://doi.org/10.1385/1-59745-037-5:233.
Velkey JM, Slawny NA, Gratsch TE, O’Shea KS. Gene silencing using RNA interference in embryonic stem cells. Methods Mol Biol. 2006;329:233–61.
Velkey, J. Matthew, et al. “Gene silencing using RNA interference in embryonic stem cells.Methods Mol Biol, vol. 329, 2006, pp. 233–61. Pubmed, doi:10.1385/1-59745-037-5:233.
Velkey JM, Slawny NA, Gratsch TE, O’Shea KS. Gene silencing using RNA interference in embryonic stem cells. Methods Mol Biol. 2006;329:233–261.

Published In

Methods Mol Biol

DOI

ISSN

1064-3745

Publication Date

2006

Volume

329

Start / End Page

233 / 261

Location

United States

Related Subject Headings

  • Transfection
  • RNA, Small Interfering
  • RNA Interference
  • Pluripotent Stem Cells
  • Plasmids
  • MicroRNAs
  • Mice
  • Liposomes
  • Lipids
  • Immunohistochemistry