Center for Research in Transplantation and Translational Immunology

GenoCellEdit

GENOME EDITING PLATFORM WITH CRISPR-Cas

The GenoCellEdit platform (PF-GCE) is associated to team 2 of the UMR INSERM 1064-CRTI. PF-GCE promotes genome editing technology, in particular through the CRISPR/Cas system. Since June 2014, it has been open to academic researchers from INSERM 1064-CRTI. Its R&D activities support innovative projects aimed at advancing technology and its applications. GCE is under the direction of Ignacio Anegon and Jean-Marie Heslan (IR) is the Technical Authority.

Technology

Artificials nucleases consist primarily of ZFNs, Meganucleases, Tales Nucleases and CRISPR/Cas. These three types of nucleases recognize specific sequences and generate DNA breaks through different mechanisms, but DNA repair pathways are common: NHEJ repair (non-homologous end joining) or homologous recombination in the presence of donor DNA. The NHEJ is a mechanism conducive to errors during repair which generates deletions and/or nucleotide insertions that can produce reading frame shifts causing gene invalidation (knock-out or KO). Homologous recombination allows new sequences to be introduced into the target gene (knock-in or KI). Thus, these artificial nucleases allow to generate both KO and/or KI.

Some applications

  • Gene Invalidation (Knock-out)
  • Insertion of exogenous DNA into a specific genomic locus (KI)
  • Insertion of a specific mutation
  • Genome modification of different species (e.g., human cells, mice, rats, etc.)
  • Gene marking (KI of a fluorescent reporter gene)
  • Genome editing in IPS cells
  • Resistance to infectious agents
  • Gene therapy: targeted integration, gene repair

Know-how

  • In silico design of guide RNA targeting the genomic site of interest
  • In cellulo functional validation of guide RNA
  • DNA donor design to modify the genome and its detection
  • Tool vectorization (plasmids, RNA, viral vectors)
  • Production of mRNA
  • Advice on strategy, type of Cas9, vectors and their uses

Type of services

  • Basic service: design and validation of guide RNA, vectorization in a plasmid, advice and project support
  • Analyses of off-target activity (depending on the species)
  • DNA donor design and construction
  • Vectorization as synthetic RNAs or in an all-in-1 recombinant viral vector (adenoviral vectors, lentiviral vectors)
  • Technological training in the detection of induced mutations
  • Custom service: depending on the user’s wishes, tasks can be shared with the platform for optimal use of the respective resources
  • Research and development

Publications

2021

2020

2019

  • Ceruloplasmin Deficiency Does Not Induce Macrophagic Iron Overload: Lessons from a New Rat Model of Hereditary Aceruloplasminemia. FASEB Journal: Official Publication of the Federation of American Societies for Experimental Biology, septembre, fj201901106R. https://doi.org/10.1096/fj.201901106R
  • Characterization of Kcnk3-Mutated Rat, a Novel Model of Pulmonary Hypertension. Circulation Research 125 (7): 678‑95. https://doi.org/10.1161/CIRCRESAHA.119.314793
  • Immunophenotype of a Rat Model of Duchenne’s Disease and Demonstration of Improved Muscle Strength After Anti-CD45RC Antibody Treatment. Frontiers in Immunology 10: 2131. https://doi.org/10.3389/fimmu.2019.02131

2018

2017

2016

  • A Rapid and Cost-Effective Method for Genotyping Genome-Edited Animals: A Heteroduplex Mobility Assay Using Microfluidic Capillary Electrophoresis. Journal of Genetics and Genomics = Yi Chuan Xue Bao, 43(5), 341‑348. https://doi.org/10.1016/j.jgg.2016.04.005
  • Pronuclear injection for the production of transgenic rats. Cold Spring Harbor protocols.
  • Comparative Analysis of piggyBac, CRISPR/Cas9 and TALEN Mediated BAC Transgenesis in the Zygote for the Generation of Humanized SIRPA Rats. Scientific Reports, 6, 31455. https://doi.org/10.1038/srep31455
  • IL-22BP is produced by eosinophils in human gut and blocks IL-22 protective actions during colitis. Mucosal Immunology, 9(2), 539‑549. https://doi.org/10.1038/mi.2015.83
  • New insights and current tools for genetically engineered (GE) sheep and goats. Theriogenology, 86(1), 160‑169. https://doi.org/10.1016/j.theriogenology.2016.04.028
  • Improved Genome Editing Efficiency and Flexibility Using Modified Oligonucleotides with TALEN and CRISPR-Cas9 Nucleases. Cell Reports, 14(9), 2263‑2272. https://doi.org/10.1016/j.celrep.2016.02.018
  • Genome Editing in Rats Using TALE Nucleases. Methods in Molecular Biology (Clifton, N.J.), 1338, 245‑259. https://doi.org/10.1007/978-1-4939-2932-0_18

2015

2014

2013

  • Human antibody expression in transgenic rats: comparison of chimeric IgH loci with human VH, D and JH but bearing different rat C-gene regions. Journal of Immunological Methods, 400401, 78‑86. https://doi.org/10.1016/j.jim.2013.10.007
  • Generation of Rag1-knockout immunodeficient rats and mice using engineered meganucleases. FASEB Journal: Official Publication of the Federation of American Societies for Experimental Biology, 27(2), 703‑711. https://doi.org/10.1096/fj.12-219907
  • Technical advances in the generation of transgenic animals and in their applications. Nantes, France, June 7th 2013. Transgenic Research, 22(5), 1065‑1072. https://doi.org/10.1007/s11248-013-9736-1
  • High-affinity IgG antibodies develop naturally in Ig-knockout rats carrying germline human IgH/Igκ/Igλ loci bearing the rat CH region. Journal of Immunology (Baltimore, Md.: 1950), 190(4), 1481‑1490. https://doi.org/10.4049/jimmunol.1203041

2012

2011

  • Effects of BCL-2 over-expression on B cells in transgenic rats and rat hybridomas. International Immunology, 23(10), 625‑636. https://doi.org/10.1093/intimm/dxr071
  • Generation of Transgenic Rats Using Microinjection of Plasmid DNA or Lentiviral Vectors. In S. Pease & T. L. Saunders (Éd.), Advanced Protocols for Animal Transgenesis: An ISTT Manual (p. 117‑135). Berlin, Heidelberg: Springer Berlin Heidelberg. https://doi.org/10.1007/978-3-642-20792-1_7
  • Knockout rats generated by embryo microinjection of TALENs. Nature Biotechnology, 29(8), 695‑696. https://doi.org/10.1038/nbt.1940

2010

2009

2005

  • Transgenic Modifications of the Rat Genome. Transgenic Research, 14(5), 531–546. https://doi.org/10.1007/s11248-005-5077-z
  • Transgenic expression of CTLA4-Ig by fetal pig neurons for xenotransplantation. Transgenic Research, 14(4), 373–384.
  • Vascular beta-adrenergic remodeling in rat transgenic model over-expressing endothelial beta3-adrenoceptors. Archives Des Maladies Du Coeur Et Des Vaisseaux, 98(7–8), 836–840.

2002-2003

  • Generation of heme oxygenase-1-transgenic rats. Experimental Biology and Medicine (Maywood, N.J.), 228(5), 466–471.
  • No functional benefit for hDAF-transgenic rat livers despite protection from tissue damage following perfusion with human serum. Transplant International: Official Journal of the European Society for Organ Transplantation, 15(12), 595–601. https://doi.org/10.1007/s00147-002-0463-4
  • Differential sensitivity of endothelial cells of various species to apoptosis induced by gene transfer of Fas ligand: Role of FLIP levels. Molecular Medicine (Cambridge, Mass.), 8(10), 612–623.
  • lacZ transgenic rats tolerant for beta-galactosidase: Recipients for gene transfer studies using lacZ as a reporter gene. Human Gene Therapy, 13(11), 1383–1390. https://doi.org/10.1089/104303402760128603
  • Cytotoxic immune response blunts long-term transgene expression after efficient retroviral-mediated hepatic gene transfer in rat. Molecular Therapy: The Journal of the American Society of Gene Therapy, 5(4), 388–396. https://doi.org/10.1006/mthe.2002.0561
  • Rapid and accurate determination of zygosity in transgenic animals by real-time quantitative PCR. Transgenic Research, 11(1), 43–48.

1999-2001

  • Cellular immunity overrules the protective effect of human DAF as demonstrated in an ex vivo heart perfusion model. Transplantation Proceedings, 33(1–2), 781–782.
  • Cryopreservation procedure for 1-cell and two-cell stage transgenic rat embryos. Transgenics, 3, 237–242.
  • Protection against hyperacute xenograft rejection of transgenic rat hearts expressing human decay accelerating factor (DAF) transplanted into primates. Molecular Medicine (Cambridge, Mass.), 5(9), 617–630.
  • Optimization of cryopreservation procedures for rat embryos. Transplantation Proceedings, 31(3), 1531–1532.
  • Endothelial expression of Fas ligand in transgenic rats under the temporal control of a tetracycline-inducible system. Transplantation Proceedings, 31(3), 1533–1534.

1996-1997

  • Production of transgenic rats for human regulators of complement activation. Transplantation Proceedings, 29(3), 1770.
  • Generation and use of transgenic rats. Rat Genome, 3, 125–132.
  • of human CD59 tissue expression directed by the CMV-IE-1 promoter in transgenic rats. Transgenic Research, 5(6), 443–450.
  • Transgenesis in rats: Technical aspects and models. Transgenic Research, 5(4), 223–234.
These services are associated with the TRIP platform (Responsable I. Anegon) and the iPS platform (Responsable L. David) for rat transgenesis and iPS cells, respectively.

Mis à jour le 21 January 2022.
https://cr2ti.univ-nantes.fr/research/equipments-core-facilities/genocelledit