Abstract
One of the great advantages of embryonic stem (ES) cells over other cell types is their accessibility to genetic manipulation. They can easily undergo genetic modifications while remaining pluripotent, and can be selectively propagated, allowing the clonal expansion of genetically altered cells in culture. Since the first isolation of ES cells in mice, many effective techniques have been developed for gene delivery and manipulation of ES cells. These include transfection, electroporation, and infection protocols, as well as different approaches for inserting, deleting, or changing the expression of genes. These methods proved to be extremely useful in mouse ES cells, for monitoring and directing differentiation, discovering unknown genes, and studying their function, and are now being extensively implemented in human ES cells (HESCs). This chapter describes the different approaches and methodologies that have been applied for the genetic manipulation of HESCs and their applications. Detailed protocols for generating clones of genetically modified HESCs by transfection, electroporation, and infection will be described, with special emphasis on the important technical details that are required for this purpose. All protocols are equally effective in human-induced pluripotent stem (iPS) cells.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Eiges R, Schuldiner M, Drukker M, Yanuka O, Itskovitz-Eldor J, Benvenisty N (2001) Establishment of human embryonic stem cell-transfected clones carrying a marker for undifferentiated cells. Curr Biol 11:514–518
Gerrard L, Zhao D, Clark AJ, Cui W (2005) Stably transfected human embryonic stem cell clones express OCT4-specific green fluorescent protein and maintain self-renewal and pluripotency. Stem Cells 23:124–133
Huber I, Itzhaki I, Caspi O, Arbel G, Tzukerman M, Gepstein A, Habib M, Yankelson L, Kehat I, Gepstein L (2007) Identification and selection of cardiomyocytes during human embryonic stem cell differentiation. FASEB J 21:2551–2563
Klug MG, Soonpaa MH, Koh GY, Field LJ (1996) Genetically selected cardiomyocytes from differentiating embryonic stem cells form stable intracardiac grafts. J Clin Invest 98:216–224
Lavon N, Yanuka O, Benvenisty N (2004) Differentiation and isolation of hepatic-like cells from human embryonic stem cells. Differentiation 72:230–238
Lavon N, Yanuka O, Benvenisty N (2006) The effect of overexpression of Pdx1 and Foxa2 on the differentiation of human embryonic stem cells into pancreatic cells. Stem Cells 24:1923–1930
Li M, Pevny L, Lovell-Badge R, Smith A (1998) Generation of purified neural precursors from embryonic stem cells by lineage selection. Curr Biol 8:971–974
Singh Roy N, Nakano T, Xuing L, Kang J, Nedergaard M, Goldman SA (2005) Enhancer-specified GFP-based FACS purification of human spinal motor neurons from embryonic stem cells. Exp Neurol 196:224–234
Bowles KM, Vallier L, Smith JR, Alexander MR, Pedersen RA (2006) HOXB4 overexpression promotes hematopoietic development by human embryonic stem cells. Stem Cells 24:1359–1369
Dekel I, Magal Y, Pearson-White S, Emerson CP, Shani M (1992) Conditional conversion of ES cells to skeletal muscle by an exogenous MyoD1 gene. New Biol 4:217–224
Kim JH, Auerbach JM, Rodriguez-Gomez JA, Velasco I, Gavin D, Lumelsky N, Lee SH, Nguyen J, Sanchez-Pernaute R, Bankiewicz K, McKay R (2002) Dopamine neurons derived from embryonic stem cells function in an animal model of Parkinson’s disease. Nature 418:50–56
Levinson-Dushnik M, Benvenisty N (1997) Involvement of hepatocyte nuclear factor 3 in endoderm differentiation of embryonic stem cells. Mol Cell Biol 17:3817–3822
Soria B, Roche E, Berna G, Leon-Quinto T, Reig JA, Martin F (2000) Insulin-secreting cells derived from embryonic stem cells normalize glycemia in streptozotocin-induced diabetic mice. Diabetes 49:157–162
Costa M, Dottori M, Sourris K, Jamshidi P, Hatzistavrou T, Davis R, Azzola L, Jackson S, Lim SM, Pera M, Elefanty AG, Stanley EG (2007) A method for genetic modification of human embryonic stem cells using electroporation. Nat Protoc 2:792–796
Hay DC, Sutherland L, Clark J, Burdon T (2004) Oct-4 knockdown induces similar patterns of endoderm and trophoblast differentiation markers in human and mouse embryonic stem cells. Stem Cells 22:225–235
Stanford WL, Cohn JB, Cordes SP (2001) Gene-trap mutagenesis: past, present and beyond. Nat Rev Genet 2:756–768
Guo G, Huang Y, Humphreys P, Wang X, Smith A (2011) A PiggyBac-based recessive screening method to identify pluripotency regulators. PLoS One 6:e18189
Capecchi MR (1989) Altering the genome by homologous recombination. Science 244:1288–1292
Urbach A, Schuldiner M, Benvenisty N (2004) Modeling for Lesch-Nyhan disease by gene targeting in human embryonic stem cells. Stem Cells 22:635–641
Davis RP, Ng ES, Costa M, Mossman AK, Sourris K, Elefanty AG, Stanley EG (2008) Targeting a GFP reporter gene to the MIXL1 locus of human embryonic stem cells identifies human primitive streak-like cells and enables isolation of primitive hematopoietic precursors. Blood 111:1876–1884
Zwaka TP, Thomson JA (2003) Homologous recombination in human embryonic stem cells. Nat Biotechnol 21:319–321
Ding Q, Regan SN, Xia Y, Oostrom LA, Cowan CA, Musunuru K (2013) Enhanced efficiency of human pluripotent stem cell genome editing through replacing TALENs with CRISPRs. Cell Stem Cell 12:393–394
Hockemeyer D, Soldner F, Beard C, Gao Q, Mitalipova M, DeKelver RC, Katibah GE, Amora R, Boydston EA, Zeitler B, Meng X, Miller JC, Zhang L, Rebar EJ, Gregory PD, Urnov FD, Jaenisch R (2009) Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases. Nat Biotechnol 27:851–857
Kiskinis E, Sandoe J, Williams LA, Boulting GL, Moccia R, Wainger BJ, Han S, Peng T, Thams S, Mikkilineni S, Mellin C, Merkle FT, Davis-Dusenbery BN, Ziller M, Oakley D, Ichida J, Di Costanzo S, Atwater N, Maeder ML, Goodwin MJ, Nemesh J, Handsaker RE, Paull D, Noggle S, McCarroll SA, Joung JK, Woolf CJ, Brown RH, Eggan K (2014) Pathways disrupted in human ALS motor neurons identified through genetic correction of mutant SOD1. Cell Stem Cell 14:781–795
Liu GH, Suzuki K, Qu J, Sancho-Martinez I, Yi F, Li M, Kumar S, Nivet E, Kim J, Soligalla RD, Dubova I, Goebl A, Plongthongkum N, Fung HL, Zhang K, Loring JF, Laurent LC, Izpisua Belmonte JC (2011) Targeted gene correction of laminopathy-associated LMNA mutations in patient-specific iPSCs. Cell Stem Cell 8:688–694
Smith-Arica JR, Thomson AJ, Ansell R, Chiorini J, Davidson B, McWhir J (2003) Infection efficiency of human and mouse embryonic stem cells using adenoviral and adeno-associated viral vectors. Cloning Stem Cells 5:51–62
Soldner F, Laganiere J, Cheng AW, Hockemeyer D, Gao Q, Alagappan R, Khurana V, Golbe LI, Myers RH, Lindquist S, Zhang L, Guschin D, Fong LK, Vu BJ, Meng X, Urnov FD, Rebar EJ, Gregory PD, Zhang HS, Jaenisch R (2011) Generation of isogenic pluripotent stem cells differing exclusively at two early onset Parkinson point mutations. Cell 146:318–331
Kim H, Kim JS (2014) A guide to genome engineering with programmable nucleases. Nat Rev Genet 15:321–334
Li M, Suzuki K, Kim NY, Liu GH, Izpisua Belmonte JC (2014) A cut above the rest: targeted genome editing technologies in human pluripotent stem cells. J Biol Chem 289:4594–4599
Vallier L, Rugg-Gunn PJ, Bouhon IA, Andersson FK, Sadler AJ, Pedersen RA (2004) Enhancing and diminishing gene function in human embryonic stem cells. Stem Cells 22:2–11
Gropp M, Itsykson P, Singer O, Ben-Hur T, Reinhartz E, Galun E, Reubinoff BE (2003) Stable genetic modification of human embryonic stem cells by lentiviral vectors. Mol Ther 7:281–287
Ma Y, Ramezani A, Lewis R, Hawley RG, Thomson JA (2003) High-level sustained transgene expression in human embryonic stem cells using lentiviral vectors. Stem Cells 21:111–117
Pfeifer A, Ikawa M, Dayn Y, Verma IM (2002) Transgenesis by lentiviral vectors: lack of gene silencing in mammalian embryonic stem cells and preimplantation embryos. Proc Natl Acad Sci U S A 99:2140–2145
Park JH, Kim SJ, Oh EJ, Moon SY, Roh SI, Kim CG, Yoon HS (2003) Establishment and maintenance of human embryonic stem cells on STO, a permanently growing cell line. Biol Reprod 69:2007–2014
Richards M, Fong CY, Chan WK, Wong PC, Bongso A (2002) Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells. Nat Biotechnol 20:933–936
Amit M, Margulets V, Segev H, Shariki K, Laevsky I, Coleman R, Itskovitz-Eldor J (2003) Human feeder layers for human embryonic stem cells. Biol Reprod 68:2150–2156
Hovatta O, Mikkola M, Gertow K, Stromberg AM, Inzunza J, Hreinsson J, Rozell B, Blennow E, Andang M, Ahrlund-Richter L (2003) A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells. Hum Reprod 18:1404–1409
Cheng L, Hammond H, Ye Z, Zhan X, Dravid G (2003) Human adult marrow cells support prolonged expansion of human embryonic stem cells in culture. Stem Cells 21:131–142
Reubinoff BE, Pera MF, Fong CY, Trounson A, Bongso A (2000) Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol 18:399–404
Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM (1998) Embryonic stem cell lines derived from human blastocysts. Science 282:1145–1147
Tucker KL, Wang Y, Dausman J, Jaenisch R (1997) A transgenic mouse strain expressing four drug-selectable marker genes. Nucleic Acids Res 25:3745–3746
Robertson EJ (1987) Teratocarcinomas and embryonic stem cells: a practical approach. IRL Press, Oxford
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media New York
About this protocol
Cite this protocol
Eiges, R. (2014). Genetic Manipulation of Human Embryonic Stem Cells. In: Turksen, K. (eds) Human Embryonic Stem Cell Protocols. Methods in Molecular Biology, vol 1307. Humana Press, New York, NY. https://doi.org/10.1007/7651_2014_155
Download citation
DOI: https://doi.org/10.1007/7651_2014_155
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2667-1
Online ISBN: 978-1-4939-2668-8
eBook Packages: Springer Protocols