title: Direct introduction of gene constructs into the pronucleus-like structure of cloned embryos: a new strategy for the generation of genetically modified pigs.
abstract: Due to a rising demand of porcine models with complex genetic modifications for biomedical research, the approaches for their generation need to be adapted. In this study we describe the direct introduction of a gene construct into the pronucleus (PN)-like structure of cloned embryos as a novel strategy for the generation of genetically modified pigs, termed "nuclear injection". To evaluate the reliability of this new strategy, the developmental ability of embryos in vitro and in vivo as well as the integration and expression efficiency of a transgene carrying green fluorescence protein (GFP) were examined. Eighty percent of the cloned pig embryos (633/787) exhibited a PN-like structure, which met the prerequisite to technically perform the new method. GFP fluorescence was observed in about half of the total blastocysts (21/40, 52.5%), which was comparable to classical zygote PN injection (28/41, 68.3%). In total, 478 cloned embryos injected with the GFP construct were transferred into 4 recipients and from one recipient 4 fetuses (day 68) were collected. In one of the fetuses which showed normal development, the integration of the transgene was confirmed by PCR in different tissues and organs from all three primary germ layers and placenta. The integration pattern of the transgene was mosaic (48 out of 84 single-cell colonies established from a kidney were positive for GFP DNA by PCR). Direct GFP fluorescence was observed macro- and microscopically in the fetus. Our novel strategy could be useful particularly for the generation of pigs with complex genetic modifications.
authors.affiliation: Chair for Molecular Animal Breeding and Biotechnology, Center for Innovative Medical Models (CiMM), LMU Munich, Hackerstr. 27, 85764, Oberschleißheim, Germany. M.Kurome@gen.vetmed.uni-muenchen.de.
authors.forename: Mayuko
authors.identifier: 0000-0002-2725-8613
authors.initials: M
authors.lastname: Kurome
authors.affiliation: Chair for Molecular Animal Breeding and Biotechnology, Center for Innovative Medical Models (CiMM), LMU Munich, Hackerstr. 27, 85764, Oberschleißheim, Germany.
authors.forename: Simon
authors.initials: S
authors.lastname: Leuchs
authors.affiliation: Chair for Molecular Animal Breeding and Biotechnology, Center for Innovative Medical Models (CiMM), LMU Munich, Hackerstr. 27, 85764, Oberschleißheim, Germany.
authors.forename: Barbara
authors.initials: B
authors.lastname: Kessler
authors.affiliation: Chair for Molecular Animal Breeding and Biotechnology, Center for Innovative Medical Models (CiMM), LMU Munich, Hackerstr. 27, 85764, Oberschleißheim, Germany.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Kemter
authors.affiliation: Chair for Molecular Animal Breeding and Biotechnology, Center for Innovative Medical Models (CiMM), LMU Munich, Hackerstr. 27, 85764, Oberschleißheim, Germany.
authors.forename: Eva-Maria
authors.initials: EM
authors.lastname: Jemiller
authors.affiliation: Chair for Molecular Animal Breeding and Biotechnology, Center for Innovative Medical Models (CiMM), LMU Munich, Hackerstr. 27, 85764, Oberschleißheim, Germany.
authors.forename: Beatrix
authors.initials: B
authors.lastname: Foerster
authors.affiliation: Chair for Molecular Animal Breeding and Biotechnology, Center for Innovative Medical Models (CiMM), LMU Munich, Hackerstr. 27, 85764, Oberschleißheim, Germany.
authors.forename: Nikolai
authors.initials: N
authors.lastname: Klymiuk
authors.affiliation: Chair for Molecular Animal Breeding and Biotechnology, Center for Innovative Medical Models (CiMM), LMU Munich, Hackerstr. 27, 85764, Oberschleißheim, Germany.
authors.forename: Valeri
authors.initials: V
authors.lastname: Zakhartchenko
authors.affiliation: Chair for Molecular Animal Breeding and Biotechnology, Center for Innovative Medical Models (CiMM), LMU Munich, Hackerstr. 27, 85764, Oberschleißheim, Germany.
authors.forename: Eckhard
authors.initials: E
authors.lastname: Wolf
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics* / Q000254:growth & development; D002467:Cell Nucleus / Q000235:genetics*; D019976:Cloning, Organism / Q000379:methods; D004247:DNA / Q000235:genetics; D004624:Embryo Transfer / Q000379:methods; D049452:Green Fluorescent Proteins / Q000235:genetics; D053652:Nuclear Transfer Techniques; D013552:Swine; D019076:Transgenes / Q000235:genetics*; D015053:Zygote / Q000254:growth & development / Q000378:metabolism
chemical_list: D049452:Green Fluorescent Proteins; D004247:DNA
keywords: Large-size DNA constructs; Microinjection; Multiple transgenic pigs; Pronucleus-like structure; Somatic cell nuclear transfer