Home LiteratureArticle Details
PMID: 24304893 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

DICE, an efficient system for iterative genomic editing in human pluripotent stem cells.

Nucleic acids research ·Vol. 42 ·No. 5 ·2014-03-00 ·Pages e34

Zhu F, Gamboa M, Farruggio AP, Hippenmeyer S, Tasic B, Schüle B, Chen-Tsai Y, Calos MP

Abstract

To reveal the full potential of human pluripotent stem cells, new methods for rapid, site-specific genomic engineering are needed. Here, we describe a system for precise genetic modification of human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). We identified a novel human locus, H11, located in a safe, intergenic, transcriptionally active region of chromosome 22, as the recipient site, to provide robust, ubiquitous expression of inserted genes. Recipient cell lines were established by site-specific placement of a 'landing pad' cassette carrying attP sites for phiC31 and Bxb1 integrases at the H11 locus by spontaneous or TALEN-assisted homologous recombination. Dual integrase cassette exchange (DICE) mediated by phiC31 and Bxb1 integrases was used to insert genes of interest flanked by phiC31 and Bxb1 attB sites at the H11 locus, replacing the landing pad. This system provided complete control over content, direction and copy number of inserted genes, with a specificity of 100%. A series of genes, including mCherry and various combinations of the neural transcription factors LMX1a, FOXA2 and OTX2, were inserted in recipient cell lines derived from H9 ESC, as well as iPSC lines derived from a Parkinson's disease patient and a normal sibling control. The DICE system offers rapid, efficient and precise gene insertion in ESC and iPSC and is particularly well suited for repeated modifications of the same locus.

MeSH Terms
Animals Cell Line Cells, Cultured Chromosomes, Human, Pair 11 Embryonic Stem Cells/metabolism Gene Expression Genetic Loci Genome, Human Genomics/methods Homologous Recombination Humans Induced Pluripotent Stem Cells/metabolism Integrases/metabolism Mice Mutagenesis, Insertional/methods Pluripotent Stem Cells/metabolism Transcription Factors/genetics,metabolism
Chemicals
Transcription Factors Integrases
Authors & Affiliations
8 authors, click to expand affiliations / ORCID
Zhu Fangfang
Department of Genetics, Stanford University, Stanford, CA 94305, USA, Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, CA 94305, USA, Howard Hughes Medical Institute and Department of Biology, Stanford University, Stanford, CA 94305, USA, Parkinson's Institute and Clinical Center, Sunnyvale, CA 94085, USA and Stanford Transgenic Research Facility, Stanford University, Stanford, CA 94305, USA.
Gamboa Matthew
Farruggio Alfonso P
Hippenmeyer Simon
Tasic Bosiljka
Schüle Birgitt
Chen-Tsai Yanru
Calos Michele P
References (36)
36 references, click to expand
  1. Reprogramming of murine and human somatic cells using a single polycistronic vector.
    Proc Natl Acad Sci U S A. 2009 Jan 6;106(1):157-62 PMID: 19109433
  2. Efficient production of mesencephalic dopamine neurons by Lmx1a expression in embryonic stem cells.
    Proc Natl Acad Sci U S A. 2009 May 5;106(18):7613-8 PMID: 19383789
  3. Parkinson's disease: genetics and pathogenesis.
    Annu Rev Pathol. 2011;6:193-222 PMID: 21034221
  4. Homologous recombination in human embryonic stem cells: a tool for advancing cell therapy and understanding and treating human disease.
    Clin Transl Sci. 2011 Aug;4(4):298-305 PMID: 21884519
  5. Site-specific cassette exchange and germline transmission with mouse ES cells expressing phiC31 integrase.
    Nat Biotechnol. 2003 Mar;21(3):321-4 PMID: 12563279
  6. ΦC31 integrase mediates efficient site-specific integration in sheep fibroblasts.
    Biosci Biotechnol Biochem. 2012;76(11):2093-5 PMID: 23132571
  7. Foxa1 and Foxa2 function both upstream of and cooperatively with Lmx1a and Lmx1b in a feedforward loop promoting mesodiencephalic dopaminergic neuron development.
    Dev Biol. 2009 Sep 15;333(2):386-96 PMID: 19607821
  8. A comparison of exogenous promoter activity at the ROSA26 locus using a ΦiC31 integrase mediated cassette exchange approach in mouse ES cells.
    PLoS One. 2011;6(8):e23376 PMID: 21853122
  9. Genetic mosaic dissection of Lis1 and Ndel1 in neuronal migration.
    Neuron. 2010 Nov 18;68(4):695-709 PMID: 21092859
  10. RNA-guided human genome engineering via Cas9.
    Science. 2013 Feb 15;339(6121):823-6 PMID: 23287722
  11. Wnt1-lmx1a forms a novel autoregulatory loop and controls midbrain dopaminergic differentiation synergistically with the SHH-FoxA2 pathway.
    Cell Stem Cell. 2009 Dec 4;5(6):646-58 PMID: 19951692
  12. Breaking the code of DNA binding specificity of TAL-type III effectors.
    Science. 2009 Dec 11;326(5959):1509-12 PMID: 19933107
  13. Correction of multi-gene deficiency in vivo using a single 'self-cleaving' 2A peptide-based retroviral vector.
    Nat Biotechnol. 2004 May;22(5):589-94 PMID: 15064769
  14. Site-specific transformation of Drosophila via phiC31 integrase-mediated cassette exchange.
    Genetics. 2006 Jun;173(2):769-77 PMID: 16547094
  15. P[acman]: a BAC transgenic platform for targeted insertion of large DNA fragments in D. melanogaster.
    Science. 2006 Dec 15;314(5806):1747-51 PMID: 17138868
  16. Find and replace: editing human genome in pluripotent stem cells.
    Protein Cell. 2011 Dec;2(12):950-6 PMID: 22173708
  17. A diversity of serine phage integrases mediate site-specific recombination in mammalian cells.
    Mol Genet Genomics. 2006 Aug;276(2):135-46 PMID: 16699779
  18. Highly efficient site-specific transgenesis in cancer cell lines.
    Mol Cancer. 2012 Dec 11;11:89 PMID: 23231822
  19. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting.
    Nucleic Acids Res. 2011 Jul;39(12):e82 PMID: 21493687
  20. Extensions of MADM (mosaic analysis with double markers) in mice.
    PLoS One. 2012;7(3):e33332 PMID: 22479386
  21. TAL Effector-Nucleotide Targeter (TALE-NT) 2.0: tools for TAL effector design and target prediction.
    Nucleic Acids Res. 2012 Jul;40(Web Server issue):W117-22 PMID: 22693217
  22. Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases.
    Nat Biotechnol. 2009 Sep;27(9):851-7 PMID: 19680244
  23. Rapid derivation of genetically related mutants from embryonic cells harboring a recombinase-specific Trp53 platform.
    Cell Cycle. 2011 Apr 15;10(8):1261-70 PMID: 21445009
  24. Mechanisms of site-specific recombination.
    Annu Rev Biochem. 2006;75:567-605 PMID: 16756503
  25. Integration specificity of phage phiC31 integrase in the human genome.
    J Mol Biol. 2006 Mar 17;357(1):28-48 PMID: 16414067
  26. Efficient reversal of phiC31 integrase recombination in mammalian cells.
    Biotechnol J. 2012 Nov;7(11):1332-6 PMID: 22933343
  27. Identification of pseudo attP sites for phage phiC31 integrase in bovine genome.
    Biochem Biophys Res Commun. 2006 Jul 7;345(3):984-8 PMID: 16712792
  28. Gene insertion and replacement in Schizosaccharomyces pombe mediated by the Streptomyces bacteriophage phiC31 site-specific recombination system.
    Mol Genet Genomics. 2001 Aug;265(6):1031-8 PMID: 11523775
  29. A phage integrase directs efficient site-specific integration in human cells.
    Proc Natl Acad Sci U S A. 2000 May 23;97(11):5995-6000 PMID: 10801973
  30. A method for producing transgenic cells using a multi-integrase system on a human artificial chromosome vector.
    PLoS One. 2011 Feb 24;6(2):e17267 PMID: 21390305
  31. SNCA triplication Parkinson's patient's iPSC-derived DA neurons accumulate α-synuclein and are susceptible to oxidative stress.
    PLoS One. 2011;6(11):e26159 PMID: 22110584
  32. Site-specific genomic integration produces therapeutic Factor IX levels in mice.
    Nat Biotechnol. 2002 Nov;20(11):1124-8 PMID: 12379870
  33. Site-specific integrase-mediated transgenesis in mice via pronuclear injection.
    Proc Natl Acad Sci U S A. 2011 May 10;108(19):7902-7 PMID: 21464299
  34. The transcription factor orthodenticle homeobox 2 influences axonal projections and vulnerability of midbrain dopaminergic neurons.
    Brain. 2010 Jul;133(Pt 7):2022-31 PMID: 20573704
  35. Genetic engineering of human pluripotent cells using TALE nucleases.
    Nat Biotechnol. 2011 Jul 07;29(8):731-4 PMID: 21738127
  36. Phage Bxb1 integrase mediates highly efficient site-specific recombination in mammalian cells.
    Biotechniques. 2006 Apr;40(4):460, 462, 464 PMID: 16629393
Article Info
Journal
Nucleic acids research
Abbr.
Nucleic Acids Res
ISSN
1362-4962
DOI
10.1093/nar/gkt1290
Published
2014-03-00
Epub
2013-00-04
Pages
e34
Language
English
Region
England
NLM ID
0411011
PMCID
PMC3950688
Subset
IM
External Links
PubMed source DOI: 10.1093/nar/gkt1290 PMC Full Text View journal details
Analysis Services
Analysis Services

Contact

No. 2 Wenbo Road, Zhangqiu District, Jinan, Shandong

Qilu Normal University · Genelibs Bioinformatics Lab

750 Shunhua Rd, Jinan

2F, Bldg F, University Science Park

Tel: 0531-88819269

WeChat Official Account

Follow our WeChat subscription account for real-time updates and the latest in medical and biological research.


Business Email

E-mail: [email protected]

Copyright © 2026.Genelibs Jinan Honghui Biotechnology Co., Ltd. 鲁ICP备20007159号-2