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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.
 Science Education: Essentials of Genetics

Recombineering and Gene Targeting

JoVE Science Education

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

 JoVE 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

 JoVE Developmental Biology

Technique to Target Microinjection to the Developing Xenopus Kidney

1Department of Pediatrics, Pediatric Research Center, University of Texas McGovern Medical School, 2Program in Genes & Development, University of Texas Graduate School of Biomedical Sciences, 3Program in Cell & Regulatory Biology, University of Texas Graduate School of Biomedical Sciences, 4Department of Genetics, University of Texas MD Anderson Cancer Center


JoVE 53799

 JoVE 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

 JoVE Genetics

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

 JoVE Neuroscience

Functional Interrogation of Adult Hypothalamic Neurogenesis with Focal Radiological Inhibition

1Division of Biology, California Institute of Technology, 2Solomon H. Snyder Department of Neuroscience, Neurology, and Ophthalamology, Johns Hopkins University School of Medicine, 3Department of Radiation Oncology & Molecular Radiation Sciences, Johns Hopkins University School of Medicine, 4Department of Radiation Oncology, University Of Washington Medical Center, 5Institute for Cell Engineering and High-Throughput Biology Center, Johns Hopkins University School of Medicine


JoVE 50716

 JoVE Bioengineering

Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles

1Division of Cardiovascular Diseases, Mayo Clinic, 2Division of Engineering, Mayo Clinic, 3School of Medicine, Pharmacy and Health, Durham University, 4Regional Center for Applied Molecular Oncology, Masaryk Memorial Cancer Institute, 5Mayo Clinic College of Medicine, Mayo Clinic


JoVE 53099

 JoVE Medicine

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

1Tumor Microenvironment and Metastasis Program, Wistar Institute, 2Department of Pathology and Lab Medicine, Perelman School of Medicine, University of Pennsylvania, 3Department of Microbiology and Immunology and Department of Genetics, Geisel School of Medicine at Dartmouth, 4Division of Endocrine and Oncologic Surgery, Department of Surgery, Perelman School of Medicine, University of Pennsylvania, 5Rena Rowan Breast Center, Abramson Cancer Center, University of Pennsylvania, 6Center for Advanced Medicine, University of Pennsylvania


JoVE 51171

 JoVE Developmental Biology

Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos

1Department of Nanobiomedical Sciences and BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, 2Insititute of Tissue Regeneration Engineering, Dankook University, 3Department of Biological Sciences, Ajou University, 4Department of Pharmacy, Sahmyook University, 5Department of Animal Biotechnology, Sahmyook University


JoVE 53696

 JoVE Medicine

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

1Molecular Virology and Gene Therapy, KU Leuven, 2Department of Woman and Child, KU Leuven, 3Neurobiology and Gene Therapy, KU Leuven, 4Division of Nuclear Medicine, KU Leuven, 5Biomedical NMR Unit/ MoSAIC, KU Leuven


JoVE 4219

 JoVE Developmental Biology

Imaging Subcellular Structures in the Living Zebrafish Embryo

1Institute of Neuronal Cell Biology, Technische Universität München, 2Cell Biology, Department of Biology, Faculty of Science, Utrecht University, 3Faculty of Biology, Ludwig-Maximilians-Universität-München, 4Adolf-Butenandt-Institute, Biochemistry, Ludwig-Maximilians-Universität-München, 5German Center for Neurodegenerative Diseases, 6Laboratory of Brain Development and Repair, The Rockefeller University


JoVE 53456

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