High‐Efficiency Transfection and siRNA‐Mediated Gene Knockdown in Human Pluripotent Stem Cells

Yinghong Ma1, Haifan Lin2, Caihong Qiu2

1 Yale Stem Cell Center, School of Medicine, Yale University, New Haven, Connecticut, 2 Department of Cell Biology, School of Medicine, Yale University, New Haven, Connecticut
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 5C.2
DOI:  10.1002/9780470151808.sc05c02s21
Online Posting Date:  May, 2012
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Abstract

This unit describes a protocol on how to achieve high transfection efficiency on human embryonic stem cells and induced pluripotent stem cells using the common transfection reagent Lipofectamine 2000 as a carrier instead of involving a virus, and/or expensive equipment and reagents. Applying this technique for siRNA‐mediated gene targeting to knockdown genes in the pluripotent stem cells, the expression of pluripotent genes, such as OCT4 and LIN28, was downregulated by more than 90% in multiple pluripotent cell lines. Beyond reaching high transfection efficiency on pluripotent cells, this protocol should also have application to primary cells that are traditionally difficult to transfect. Curr. Protoc. Stem Cell Biol. 21:5C.2.1‐5C.2.9. © 2012 by John Wiley & Sons, Inc.

Keywords: transfection; human embryonic stem cells; induced pluripotent stem cells; siRNA gene knockdown

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Cloning
  • Basic Protocol 2: Plasmid Transfection
  • Alternate Protocol 1: siRNA‐Mediated Gene Knockdown
  • Support Protocol 1: Preparing Matrigel‐Coated Plates
  • Basic Protocol 3: Culture Pluripotent Cells on Feeder‐Free, Serum‐Free, and Component‐Defined Conditions
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Cloning

  Materials
  • hESCs and iPSCs grown on Matrigel‐coated plate under feeder‐free, serum‐free, and component‐defined conditions
  • Pluripotent cell medium (see recipe)
  • Rock inhibitor Y‐27632 (Calbiochem, cat. no. 688001)
  • DMEM/F12 medium
  • Accutase (Stemcell Technology, cat. no. 07920)
  • Matrigel‐coated 6‐well plates (see protocol 4)
  • 6‐well plates
  • 37°C incubator
  • 15‐ml Falcon tubes
  • Centrifuge

Basic Protocol 2: Plasmid Transfection

  Materials
  • Plasmid pSUPER‐GFP is a 5.4 kb vector driven by a CMV promoter (Oligoangine)
  • OPTI‐MEM
  • Lipofectamine 2000 (Invitrogen, cat. no. 11668‐019)
  • 1 million cells (see protocol 1)
  • DMEM/F12 medium
  • Matrigel‐coated 6‐well plates (see protocol 4)
  • Pluripotent cell medium (see recipe)
  • Rock inhibitor Y‐27632 (Calbiochem, cat. no. 688001)
  • 37°C, 5% CO 2, 5% O 2 incubator

Alternate Protocol 1: siRNA‐Mediated Gene Knockdown

  Materials
  • Transfection solution (see recipe)
  • siRNA targeting Oct4 (siOct4; Dharmacon, cat. no. L‐019591‐00) and control siRNA (siCon; Dharmacon, cat. no. D‐001810‐10‐05)
  • Pluripotent cell medium (see recipe)
  • 37°C, 5% CO 2, 5% O 2 incubator
  • Additional reagents and equipment for transfection (see protocol 2)

Support Protocol 1: Preparing Matrigel‐Coated Plates

  Materials
  • Matrigel basement membrane matrix, growth factor reduced (BD, cat. no. 356231)
  • DMEM/F12 basal medium
  • 6‐well plates
  • 5% CO 2, 5% O 2 and 37°C humidified incubator
  • Parafilm

Basic Protocol 3: Culture Pluripotent Cells on Feeder‐Free, Serum‐Free, and Component‐Defined Conditions

  Materials
  • Differentiated cells (see Introduction)
  • DMEM/F12 medium
  • Dispase (Stemcell Technology, cat. no. 07913)
  • Matrigel‐coated plates (see protocol 4)
  • 5% CO 2, 5% O 2 and 37°C humidified incubator
  • 15‐ml tubes (e.g., Falcon)
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Figures

Videos

Literature Cited

Literature Cited
   Bajpai, R., Lesperance, J., Kim, M., and Terskikh, A.V. 2008. Efficient propagation of single cells Accutase‐dissociated human embryonic stem cells. Mol. Reprod. Dev. 75:818‐827.
   Claassen, D.A., Desler, M.M., and Rizzino, A. 2009. ROCK inhibition enhances the recovery and growth of cryopreserved human embryonic stem cells and human induced pluripotent stem cells. Mol. Reprod. Dev. 76:722‐732.
   Hohenstein, K.A., Pyle, A.D., Chern, J.Y., Lock, L.F., and Donovan, P.J. 2008. Nucleofection mediates high‐efficiency stable gene knockdown and transgene expression in human embryonic stem cells. Stem Cells 26:1436‐1443.
   Liu, Y., Song, Z., Zhao, Y., Qin, H., Cai, J., Zhang, H., Yu, T., Jiang, S., Wang, G., Ding, M., and Deng, H. 2006. A novel chemical‐defined medium with bFGF and N2B27 supplements supports undifferentiated growth in human embryonic stem cells. Biochem. Biophys. Res. Commun. 346:131‐139.
   Ma, Y., Jin, J., Dong, C., Cheng, E., Lin, H., Huang, Y., and Qiu, C. 2010. High‐efficiency siRNA‐based gene knockdown in human embryonic stem cells. RNA. 16:2564‐2569.
   Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., and Yamanaka, S. 2007. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131:861‐872.
   Thomson, J.A., Itskovitz‐Eldor, J., Shapiro, S.S., Waknitz, M.A., Swiergiel, J.J., Marshall, V.S., and Jones, J.M. 1998. Embryonic stem cell lines derived from human blastocysts. Science 282:1145‐1147.
   Watanabe, K., Ueno, M., Kamiya, D., Nishiyama, A., Matsumura, M., Wataya, T., Takahashi, J.B., Nishikawa, S., Muguruma, K., and Sasai, Y. 2007. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat. Biotechnol. 25:681‐686.
   Xu, C., Inokuma, M.S., Denham, J., Golds, K., Kundu, P., Gold, J.D., and Carpenter, M.K. 2001. Feeder‐free growth of undifferentiated human embryonic stem cells. Nat. Biotechnol. 19:971‐974.
   Yao, S., Chen, S., Clark, J., Hao, E., Beattie, G.M., Hayek, A., and Ding, S. 2006. Long‐term self‐renewal and directed differentiation of human embryonic stem cells in chemically defined conditions. Proc. Natl. Acad. Sci. U.S.A. 103:6907‐6912.
   Yu, J., Vodyanik, M.A., Smuga‐Otto, K., Antosiewicz‐Bourget, J., Frane, J.L., Tian, S., Nie, J., Jonsdottir, G.A., Ruotti, V., Stewart, R., Slukvin, I.I., and Thomson, J.A. 2007. Induced pluripotent stem cell lines derived from human somatic cells. Science 318:1917‐1920.
   Zaehres, H., Lensch, M.W., Daheron, L., Stewart, S.A., Itskovitz‐Eldor, J., and Daley, G.Q. 2005. High‐efficiency RNA interference in human embryonic stem cells. Stem Cells 23:299‐305.
   Zhang, M., Guller, S., and Huang, Y. 2007. Method to enhance transfection efficiency of cell lines and placental fibroblasts. Placenta 28:779‐82.
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