title: Pronuclear Injection-Based Targeted Transgenesis.
abstract: Microinjection of DNA expression cassettes into fertilized zygotes has been a standard method for generating transgenic animal models. While efficient, the injected DNA integrates randomly into the genome, leading to potential disruption of endogenous genes or regulatory elements, variation in copy number, or integration into heterochromatic regions that inhibit transgene expression. A recently developed method addresses such pitfalls of traditional transgenesis by targeting the transgene to predetermined sites in the genome that can safely harbor exogenous DNA. This method, called Pronuclear Injection-based Targeted Transgenesis (PITT), employs an enzymatic transfer of exogenous DNA from a donor vector to a previously created landing-pad site in the mouse genome. DNA transfer is achieved using molecular tools such as the Cre-LoxP recombinase and PhiC31-attB/P integrase systems. Here, we provide protocols for performing PITT and an overview of the current PITT tools available to the research community. © 2016 by John Wiley & Sons, Inc.
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, Massachusetts.
authors.forename: Samantha L P
authors.initials: SLP
authors.lastname: Schilit
authors.affiliation: Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, Isehara, Kanagawa, Japan.|The Institute of Medical Sciences, Tokai University, Isehara, Kanagawa, Japan.
authors.forename: Masato
authors.initials: M
authors.lastname: Ohtsuka
authors.affiliation: Mouse Genome Engineering Core Facility, University of Nebraska Medical Center, Omaha, Nebraska.
authors.forename: Rolen M
authors.initials: RM
authors.lastname: Quadros
authors.affiliation: Mouse Genome Engineering Core Facility, University of Nebraska Medical Center, Omaha, Nebraska.|Developmental Neuroscience, Munroe Meyer Institute, University of Nebraska Medical Center, Omaha, Nebraska.
authors.forename: Channabasavaiah B
authors.initials: CB
authors.lastname: Gurumurthy
mesh_terms: D000818:Animals; D001287:Attachment Sites, Microbiological / Q000235:genetics; D002467:Cell Nucleus / Q000235:genetics*; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004622:Embryo, Mammalian / Q000166:cytology*; D005260:Female; D018390:Gene Targeting / Q000379:methods*; D018014:Gene Transfer Techniques; D016678:Genome / Q000235:genetics; D019426:Integrases / Q000378:metabolism; D008297:Male; D051379:Mice; D008845:Microinjections; D011995:Recombination, Genetic; D019076:Transgenes / Q000235:genetics
chemical_list: D004247:DNA; C045073:Cre recombinase; D019426:Integrases
keywords: Cre-LoxP recombination; PITT; PhiC31-attB/P integration; pronuclear injection; targeted transgenesis