Single site-specific integration targeting coupled with embryonic stem cell differentiation provides a high-throughput alternative to in vivo enhancer analyses

Summary Comprehensive analysis of cis-regulatory elements is key to understanding the dynamic gene regulatory networks that control embryonic development. While transgenic animals represent the gold standard assay, their generation is costly, entails significant animal usage, and in utero development complicates time-course studies. As an alternative, embryonic stem (ES) cells can readily be differentiated in a process that correlates well with developing embryos. Here, we describe a highly effective platform for enhancer assays using an Hsp68/Venus reporter cassette that targets to the Hprt locus in mouse ES cells. This platform combines the flexibility of Gateway® cloning, live cell trackability of a fluorescent reporter, low background and the advantages of single copy insertion into a defined genomic locus. We demonstrate the successful recapitulation of tissue-specific enhancer activity for two cardiac and two haematopoietic enhancers. In addition, we used this assay to dissect the functionality of the highly conserved Ets/Ets/Gata motif in the Scl+19 enhancer, which revealed that the Gata motif is not required for initiation of enhancer activity. We further confirmed that Gata2 is not required for endothelial activity of the Scl+19 enhancer using Gata2−/− Scl+19 transgenic embryos. We have therefore established a valuable toolbox to study gene regulatory networks with broad applicability.

[1]  Hani Z. Girgis,et al.  A High-Resolution Enhancer Atlas of the Developing Telencephalon , 2013, Cell.

[2]  M. Pellegrini,et al.  Scl Represses Cardiomyogenesis in Prospective Hemogenic Endothelium and Endocardium , 2012, Cell.

[3]  Nicola K. Wilson,et al.  GFI1 and GFI1B control the loss of endothelial identity of hemogenic endothelium during hematopoietic commitment. , 2012, Blood.

[4]  B. Göttgens,et al.  Deletion of the Scl +19 enhancer increases the blood stem cell compartment without affecting the formation of mature blood lineages , 2012, Experimental hematology.

[5]  A. Visel,et al.  Large-Scale Discovery of Enhancers from Human Heart Tissue , 2011, Nature Genetics.

[6]  Margherita Neri,et al.  Site-specific integration and tailoring of cassette design for sustainable gene transfer , 2011, Nature Methods.

[7]  E. Davidson.  Emerging properties of animal gene regulatory networks , 2010, Nature.

[8]  W. Ouwehand,et al.  Combinatorial transcriptional control in blood stem/progenitor cells: genome-wide analysis of ten major transcriptional regulators. , 2010, Cell stem cell.

[9]  Berthold Göttgens,et al.  Gene regulatory networks governing haematopoietic stem cell development and identity. , 2010, The International journal of developmental biology.

[10]  N. Wilson,et al.  Gfi1 Expression Is Controlled by Five Distinct Regulatory Regions Spread over 100 Kilobases, with Scl/Tal1, Gata2, PU.1, Erg, Meis1, and Runx1 Acting as Upstream Regulators in Early Hematopoietic Cells , 2010, Molecular and Cellular Biology.

[11]  R. Jaenisch,et al.  Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases , 2009, Nature Biotechnology.

[12]  V. Kouskoff,et al.  In vitro differentiation of mouse embryonic stem cells as a model of early hematopoietic development. , 2009, Methods in molecular biology.

[13]  G. Daley,et al.  Mesodermal patterning activity of SCL. , 2008, Experimental hematology.

[14]  B. Göttgens,et al.  A novel mode of enhancer evolution: the Tal1 stem cell enhancer recruited a MIR element to specifically boost its activity. , 2008, Genome research.

[15]  P. Vyas,et al.  Differential use of SCL/TAL-1 DNA-binding domain in developmental hematopoiesis. , 2008, Blood.

[16]  R. Pedersen,et al.  Robust, Persistent Transgene Expression in Human Embryonic Stem Cells Is Achieved with AAVS1‐Targeted Integration , 2008, Stem cells.

[17]  G. Keller,et al.  Identification and targeting of the ROSA26 locus in human embryonic stem cells , 2007, Nature Biotechnology.

[18]  Berthold Göttgens,et al.  Gata2, Fli1, and Scl form a recursively wired gene-regulatory circuit during early hematopoietic development , 2007, Proceedings of the National Academy of Sciences.

[19]  Daniel G. Tenen,et al.  Transcription factors in myeloid development: balancing differentiation with transformation , 2007, Nature Reviews Immunology.

[20]  M. Kyba,et al.  GATA2 functions at multiple steps in hemangioblast development and differentiation , 2007, Development.

[21]  Inna Dubchak,et al.  VISTA Enhancer Browser—a database of tissue-specific human enhancers , 2006, Nucleic Acids Res..

[22]  Alan M. Moses,et al.  In vivo enhancer analysis of human conserved non-coding sequences , 2006, Nature.

[23]  Anil Kumar.  Regulation of Stem Cell , 2006 .

[24]  J. D. Engel,et al.  Combinatorial Gata2 and Sca1 expression defines hematopoietic stem cells in the bone marrow niche , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[25]  B. Göttgens,et al.  Transgenic Analysis of the Stem Cell Leukemia +19 Stem Cell Enhancer in Adult and Embryonic Hematopoietic and Endothelial Cells , 2005, Stem cells.

[26]  S. Orkin,et al.  Gfi-1 restricts proliferation and preserves functional integrity of haematopoietic stem cells , 2004, Nature.

[27]  T. Möröy,et al.  Gfi1:Green Fluorescent Protein Knock-in Mutant Reveals Differential Expression and Autoregulation of the Growth Factor Independence 1 (Gfi1) Gene during Lymphocyte Development* , 2004, Journal of Biological Chemistry.

[28]  B. Göttgens,et al.  The scl +18/19 Stem Cell Enhancer Is Not Required for Hematopoiesis: Identification of a 5′ Bifunctional Hematopoietic-Endothelial Enhancer Bound by Fli-1 and Elf-1 , 2004, Molecular and Cellular Biology.

[29]  M. Gassmann,et al.  Differentiating embryonic stem cells into embryoid bodies. , 2004, Methods in molecular biology.

[30]  T. Lufkin,et al.  Expression of Tfap2d, the gene encoding the transcription factor Ap-2 delta, during mouse embryogenesis. , 2003, Gene expression patterns : GEP.

[31]  James A. Thomson,et al.  Homologous recombination in human embryonic stem cells , 2003, Nature Biotechnology.

[32]  S. Orkin,et al.  Expression of CD41 marks the initiation of definitive hematopoiesis in the mouse embryo. , 2003, Blood.

[33]  W. Aird,et al.  Differential regulation of the von Willebrand factor and Flt-1 promoters in the endothelium of hypoxanthine phosphoribosyltransferase-targeted mice. , 2002, Blood.

[34]  B. Göttgens,et al.  Establishing the transcriptional programme for blood: the SCL stem cell enhancer is regulated by a multiprotein complex containing Ets and GATA factors , 2002, The EMBO journal.

[35]  W. Vainchenker,et al.  Expression of CD41 on hematopoietic progenitors derived from embryonic hematopoietic cells. , 2002, Development.

[36]  Hui Zeng,et al.  Inflammatory reactions and severe neutropenia in mice lacking the transcriptional repressor Gfi1 , 2002, Nature Genetics.

[37]  K. Mikoshiba,et al.  A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications , 2002, Nature Biotechnology.

[38]  S. Bronson,et al.  Generation of single-copy transgenic mouse embryos directly from ES cells by tetraploid embryo complementation , 2001, BMC biotechnology.

[39]  A. Green,et al.  Selective rescue of early haematopoietic progenitors in Scl(-/-) mice by expressing Scl under the control of a stem cell enhancer. , 2001, Development.

[40]  Berthold Göttgens,et al.  Regulation of the stem cell leukemia (SCL) gene: A tale of two fishes , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[41]  J. Hartley,et al.  DNA cloning using in vitro site-specific recombination. , 2000, Genome research.

[42]  V. Praphanphoj,et al.  Molecular cytogenetic evaluation in a patient with a translocation (3;21) associated with blepharophimosis, ptosis, epicanthus inversus syndrome (BPES). , 2000, Genomics.

[43]  W. Aird,et al.  Targeting the Hprt locus in mice reveals differential regulation of Tie2 gene expression in the endothelium. , 2000, Physiological genomics.

[44]  B. Göttgens,et al.  An SCL 3' enhancer targets developing endothelium together with embryonic and adult haematopoietic progenitors. , 1999, Development.

[45]  N. Maeda,et al.  Single-copy transgenic mice with chosen-site integration. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[46]  C. Begley,et al.  The scl gene product is required for the generation of all hematopoietic lineages in the adult mouse. , 1996, The EMBO journal.

[47]  F. Alt,et al.  The T Cell Leukemia Oncoprotein SCL/tal-1 Is Essential for Development of All Hematopoietic Lineages , 1996, Cell.

[48]  S. Orkin,et al.  Absence of blood formation in mice lacking the T-cell leukaemia oncoprotein tal-1/SCL , 1995, Nature.

[49]  Stuart H. Orkin,et al.  An early haematopoietic defect in mice lacking the transcription factor GATA-2 , 1994, Nature.

[50]  Jürgen Hescheler,et al.  Embryonic stem cells differentiate in vitro into cardiomyocytes representing sinusnodal, atrial and ventricular cell types , 1993, Mechanisms of Development.

[51]  K. Chien,et al.  Transcriptional regulation during cardiac growth and development. , 1993, Annual review of physiology.

[52]  M. Wiles,et al.  Hematopoietic commitment during embryonic stem cell differentiation in culture. , 1993, Molecular and cellular biology.

[53]  J. Mcwhir,et al.  A new mouse embryonic stem cell line with good germ line contribution and gene targeting frequency. , 1992, Nucleic acids research.

[54]  D. Housman,et al.  Targeted integration of adeno‐associated virus (AAV) into human chromosome 19. , 1991, The EMBO journal.

[55]  S. Orkin,et al.  A nonerythroid GATA-binding protein is required for function of the human preproendothelin-1 promoter in endothelial cells , 1990, Molecular and cellular biology.

[56]  J. Rossant,et al.  Inducible expression of an hsp68-lacZ hybrid gene in transgenic mice. , 1989, Development.

[57]  A. Landy.  Dynamic, structural, and regulatory aspects of lambda site-specific recombination. , 1989, Annual review of biochemistry.