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October, 2006
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Gene Targeting: The integration of exogenous DNA into the genome of an organism at sites where its expression can be suitably controlled. This integration occurs as a result of homologous recombination.

Recombineering and Gene Targeting

JoVE 5553

One of the most widely used tools in modern biology is molecular cloning with restriction enzymes, which create compatible ends between DNA fragments that allow them to be joined together. However, this technique has certain restrictions that limit its applicability for large or complex DNA construct generation. A newer technique that addresses some of these shortcomings is recombineering, which modifies DNA using homologous recombination (HR), the exchange between different DNA molecules based on stretches of similar or identical sequences. Together with gene targeting, which takes advantage of endogenous HR to alter an organism’s genome at a specific loci, HR-based cloning techniques have greatly improved the speed and efficacy of high-throughput genetic engineering.In this video, we introduce the principles of HR, as well as the basic components required to perform a recombineering experiment, including recombination-competent organisms and genomic libraries such as bacterial artificial chromosomes (BAC). We then walk through a protocol that uses recombineering to generate a gene-targeting vector that can ultimately be transfected into embryonic stem cells to generate a transgenic animal. Finally, several applications that highlight the utility and variety of recombineering techniques wi


Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange

1Department of Biomedical Molecular Biology, Ghent University, 2Inflammation Research Center, VIB, 3Center for Medical Genetics, Ghent University Hospital, 4Cancer Research Institute Ghent (CRIG), 5Department of Basic Medical Sciences, Faculty of Medicine and Health Sciences, Ghent University, 6Helmholtz Center for Infection Research, 7Mammalian Functional Genetics Laboratory, Division of Blood Cancers, Australian Centre for Blood Diseases, Department of Clinical Haematology, Monash University and Alfred Health Alfred Centre

JoVE 55575

 Developmental Biology

Efficient Generation of hiPSC Neural Lineage Specific Knockin Reporters Using the CRISPR/Cas9 and Cas9 Double Nickase System

1Department of Neurosurgery, The University of Texas Health Science Center at Houston, 2Center for Stem Cell and Regenerative Medicine, Brown Foundation Institute of Molecular Medicine, The University of Texas Health Science Center at Houston, 3The Senator Lloyd & B. A. Bentsen Center for Stroke Research, Brown Foundation Institute of Molecular Medicine, The University of Texas Health Science Center at Houston, 4Summer Research Program, Office of Educational Programs, The University of Texas Health Science Center at Houston, 5Department of Anesthesiology, Shengjing Hospital, China Medical University, 6Department of Oncology, Renji Hospital, Shanghai Jiaotong University School of Medicine, 7Biology Department, University of West Georgia

JoVE 52539

 Developmental Biology

Chitosan/Interfering RNA Nanoparticle Mediated Gene Silencing in Disease Vector Mosquito Larvae

1Division of Biology, Kansas State University, 2Department of Medical and Molecular Genetics, Indiana University School of Medicine, 3Eck Institute for Global Health, University of Notre Dame, 4Department of Biological Sciences, University of Notre Dame, 5Department of Entomology, Kansas State University

JoVE 52523


Genetic Engineering of an Unconventional Yeast for Renewable Biofuel and Biochemical Production

1Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, 2NUS Synthetic Biology for Clinical and Technological Innovation (SynCTI), Life Sciences Institute, National University of Singapore, 3Food Science and Chemical Engineering, Singapore Institute of Technology

JoVE 54371


Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

1School of Fisheries, Aquaculture and Aquatic Sciences, Auburn University, 2Department of Animal Wealth Development, Faculty of Veterinary Medicine, Suez Canal University, 3Anatomy and Embryology Department, Faculty of Veterinary Medicine, Cairo University, 4Department of Molecular Physiology and Biophysics, Vanderbilt University, 5Life Science Institute, University of Michigan

JoVE 56275


CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

1Institute of Neuroscience, State Key Laboratory of Neuroscience, Key Laboratory of Primate Neurobiology, CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 2College of Life Sciences, University of Chinese Academy of Sciences, 3School of Life Science and Technology, Shanghai Tech University, 4Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences

JoVE 56844


Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

1Department of Molecular & Human Genetics, Baylor College of Medicine, 2Stem Cells & Regenerative Medicine Center, Baylor College of Medicine, 3Integrative Molecular and Biomedical Sciences Program, Baylor College of Medicine, 4Translational Biology and Molecular Medicine Program, Baylor College of Medicine, 5Center for Cell and Gene Therapy, Baylor College of Medicine, 6Centro di Ricerca Emato-Oncologica (CREO), University of Perugia, 7Texas Children's Hospital & Houston Methodist Hospital

Video Coming Soon

JoVE 57278

 JoVE In-Press

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

1Center for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, 2Sino-British Research Centre for Molecular Oncology, National Center for International Research in Cell and Gene Therapy, Zhengzhou University, 3School of Basic Medical Sciences, Academy of Medical Sciences, Zhengzhou University

JoVE 54171

 Immunology and Infection

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

1Division of Molecular Neurogenetics, German Cancer Research Center (DKFZ), DKFZ-ZMBH Alliance, 2Division of Molecular Genetics, German Cancer Research Center (DKFZ), and German Cancer Consortium (DKTK), 3Hopp-Children's Cancer Center at the NCT Heidelberg (KiTZ), 4Division of Pediatric Neurooncology, German Cancer Research Center (DKFZ), and German Cancer Consortium (DKTK), 5Department of Pediatric Hematology and Oncology, Heidelberg University Hospital

Video Coming Soon

JoVE 57311

 JoVE In-Press

Production of Genetically Engineered Golden Syrian Hamsters by Pronuclear Injection of the CRISPR/Cas9 Complex

1Department of Animal, Dairy, and Veterinary Sciences, Utah State University, 2National Centre for International Research in Cell and Gene Therapy, Sino-British Research Centre, School of Basic Medical Sciences, Zhengzhou University, 3Department of Animal Science Division of Applied Life Science (BK21 Plus), Gyeongsang National University, 4Institute of Agriculture and Life Science, Gyeongsang National University, 5Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London

JoVE 56263


Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

1Department of Biochemistry, University of Illinois at Urbana-Champaign, 2Department of Comparative Biosciences, University of Illinois at Urbana-Champaign, 3Neuroscience Program, University of Illinois at Urbana-Champaign, 4Center for Biophysics and Quantitative Biology, University of Illinois at Urbana-Champaign

JoVE 55823

 Developmental Biology

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