Site-specific integrase-mediated transgenesis in mice via pronuclear injection

Microinjection of recombinant DNA into zygotic pronuclei has been widely used for producing transgenic mice. However, with this method, the insertion site, integrity, and copy number of the transgene cannot be controlled. Here, we present an integrase-based approach to produce transgenic mice via pronuclear injection, whereby an intact single-copy transgene can be inserted into predetermined chromosomal loci with high efficiency (up to 40%), and faithfully transmitted through generations. We show that neighboring transgenic elements and bacterial DNA within the transgene cause profound silencing and expression variability of the transgenic marker. Removal of these undesirable elements leads to global high-level marker expression from transgenes driven by a ubiquitous promoter. We also obtained faithful marker expression from a tissue-specific promoter. The technique presented here will greatly facilitate murine transgenesis and precise structure/function dissection of mammalian gene function and regulation in vivo.

[1]  David W. Melton,et al.  Targetted correction of a mutant HPRT gene in mouse embryonic stem cells , 1987, Nature.

[2]  M. Kay,et al.  Silencing of episomal transgene expression by plasmid bacterial DNA elements in vivo , 2004, Gene Therapy.

[3]  J. Lingrel,et al.  Erythroid‐specific expression of human beta‐globin genes in transgenic mice. , 1985, The EMBO journal.

[4]  Eric C. Olivares,et al.  Site-specific genomic integration produces therapeutic Factor IX levels in mice , 2002, Nature Biotechnology.

[5]  R. Maeda,et al.  An optimized transgenesis system for Drosophila using germ-line-specific φC31 integrases , 2007, Proceedings of the National Academy of Sciences.

[6]  R. Hollis,et al.  Phage integrases for the construction and manipulation of transgenic mammals , 2003, Reproductive biology and endocrinology : RB&E.

[7]  L. Luo,et al.  Genetic Mosaic Dissection of Lis1 and Ndel1 in Neuronal Migration , 2010, Neuron.

[8]  David Garrick,et al.  Repeat-induced gene silencing in mammals , 1998, Nature Genetics.

[9]  F. Ruddle,et al.  Integration and stable germ line transmission of genes injected into mouse pronuclei. , 1981, Science.

[10]  B. Franc,et al.  Telomeric Transgenes Are Silenced in Adult Mouse Tissues and Embryo Fibroblasts but Are Expressed in Embryonic Stem Cells , 2007, Stem cells.

[11]  Michele P Calos,et al.  Construction of transgenic Drosophila by using the site-specific integrase from phage phiC31. , 2004, Genetics.

[12]  David Baltimore,et al.  Germline Transmission and Tissue-Specific Expression of Transgenes Delivered by Lentiviral Vectors , 2002, Science.

[13]  W. Wurst,et al.  Gene targeting by homologous recombination in mouse zygotes mediated by zinc-finger nucleases , 2010, Proceedings of the National Academy of Sciences.

[14]  J. Murnane,et al.  Telomere Position Effect and Silencing of Transgenes near Telomeres in the Mouse , 2006, Molecular and Cellular Biology.

[15]  H. Inoko,et al.  Pronuclear injection-based mouse targeted transgenesis for reproducible and highly efficient transgene expression , 2010, Nucleic acids research.

[16]  L. Luo,et al.  A global double‐fluorescent Cre reporter mouse , 2007, Genesis.

[17]  C. S. Raymond,et al.  High-Efficiency FLP and ΦC31 Site-Specific Recombination in Mammalian Cells , 2007, PloS one.

[18]  J. Strouboulis,et al.  Heterochromatin Effects on the Frequency and Duration of LCR-Mediated Gene Transcription , 1996, Cell.

[19]  Sheng Zhao,et al.  Comprehensive Algorithm for Quantitative Real-Time Polymerase Chain Reaction , 2005, J. Comput. Biol..

[20]  Y. Saeki,et al.  Plasmid DNA Sequences Present in Conventional Herpes Simplex Virus Amplicon Vectors Cause Rapid Transgene Silencing by Forming Inactive Chromatin , 2006, Journal of Virology.

[21]  C. Babinet,et al.  lacZ and ubiquitously expressed genes: should divorce be pronounced? , 2000, Transgenic Research.

[22]  Shankar Srinivas,et al.  Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus , 2001, BMC Developmental Biology.

[23]  M. Capecchi,et al.  Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells , 1987, Cell.

[24]  Silvia Arber,et al.  Requirement for the Homeobox Gene Hb9 in the Consolidation of Motor Neuron Identity , 1999, Neuron.

[25]  Jim Haseloff,et al.  Mutations that suppress the thermosensitivity of green fluorescent protein , 1996, Current Biology.

[26]  Hugo J. Bellen,et al.  P[acman]: A BAC Transgenic Platform for Targeted Insertion of Large DNA Fragments in D. melanogaster , 2006, Science.

[27]  D. Castrillon,et al.  Generation of a germ cell‐specific mouse transgenic Cre line, Vasa‐Cre , 2007, Genesis.

[28]  E. Fama,et al.  Migration , 2007 .

[29]  X. Cui,et al.  Targeted integration in rat and mouse embryos with zinc-finger nucleases , 2011, Nature Biotechnology.

[30]  Boris Jerchow,et al.  Molecular evolution of a novel hyperactive Sleeping Beauty transposase enables robust stable gene transfer in vertebrates , 2009, Nature Genetics.

[31]  Jason J. Hoyt,et al.  A diversity of serine phage integrases mediate site-specific recombination in mammalian cells , 2006, Molecular Genetics and Genomics.

[32]  Eric C. Olivares,et al.  A phage integrase directs efficient site-specific integration in human cells. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[33]  M. Kay,et al.  Minicircle DNA vectors devoid of bacterial DNA result in persistent and high-level transgene expression in vivo. , 2003, Molecular therapy : the journal of the American Society of Gene Therapy.

[34]  J. Roder,et al.  Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[35]  D. Ow,et al.  Site-specific cassette exchange and germline transmission with mouse ES cells expressing φC31 integrase , 2003, Nature Biotechnology.

[36]  M. Capecchi,et al.  In vivo evaluation of PhiC31 recombinase activity using a self-excision cassette , 2008, Nucleic acids research.

[37]  Philippe Soriano,et al.  Widespread recombinase expression using FLPeR (Flipper) mice , 2000, Genesis.

[38]  Philippe Soriano.  Generalized lacZ expression with the ROSA26 Cre reporter strain , 1999, Nature Genetics.

[39]  T. Ley,et al.  Targeted deletion of 5'HS2 of the murine beta-globin LCR reveals that it is not essential for proper regulation of the beta-globin locus. , 1995, Genes & development.

[40]  F H Ruddle,et al.  Genetic transformation of mouse embryos by microinjection of purified DNA. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[41]  Scott E. Fraser,et al.  Imaging Cells in the Developing Nervous System with Retrovirus Expressing Modified Green Fluorescent Protein , 1999, Experimental Neurology.

[42]  Kristina D. Micheva,et al.  Visualizing the Distribution of Synapses from Individual Neurons in the Mouse Brain , 2010, PloS one.

[43]  M. Vidal,et al.  Variegation associated with lacZ in transgenic animals: a warning note , 2000, Transgenic Research.

[44]  Margaret C. M. Smith,et al.  In vitro site-specific integration of bacteriophage DNA catalyzed by a recombinase of the resolvase/invertase family. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[45]  Min Han,et al.  Efficient Transposition of the piggyBac (PB) Transposon in Mammalian Cells and Mice , 2005, Cell.

[46]  M. Groudine,et al.  DNA cassette exchange in ES cells mediated by Flp recombinase: an efficient strategy for repeated modification of tagged loci by marker-free constructs. , 1998, Biochemistry.

[47]  S. Hughes,et al.  Altering the Genome by Homologous Recombination , 2010 .

[48]  R. Palmiter,et al.  Somatic expression of herpes thymidine kinase in mice following injection of a fusion gene into eggs , 1981, Cell.

[49]  D. Kioussis,et al.  Position effect variegation and imprinting of transgenes in lymphocytes , 2008, Nucleic Acids Research.

[50]  L. Luo,et al.  Mosaic Analysis with Double Markers in Mice , 2005, Cell.

[51]  J. Bode,et al.  Double-reciprocal crossover mediated by FLP-recombinase: a concept and an assay. , 1997, Biochemistry.

[52]  S. Pfaff,et al.  Active Suppression of Interneuron Programs within Developing Motor Neurons Revealed by Analysis of Homeodomain Factor HB9 , 1999, Neuron.