Monika Baxa1, Marian Hruska-Plochan2, Stefan Juhas3, Petr Vodicka4, Antonin Pavlok3, Jana Juhasova3, Atsushi Miyanohara5, Tetsuya Nejime6, Jiri Klima3, Monika Macakova1, Silvia Marsala7, Andreas Weiss8, Svatava Kubickova9, Petra Musilova9, Radek Vrtel10, Emily M Sontag11, Leslie M Thompson12, Jan Schier13, Hana Hansikova14, David S Howland15, Elena Cattaneo16, Marian DiFiglia17, Martin Marsala18, Jan Motlik3. 1. Laboratory of Cell Regeneration and Plasticity, Institute of Animal Physiology and Genetics, v.v.i., AS CR, Libechov, Czech Republic Faculty of Science, Department of Cell Biology, Charles University in Prague, Prague, Czech Republic. 2. Laboratory of Cell Regeneration and Plasticity, Institute of Animal Physiology and Genetics, v.v.i., AS CR, Libechov, Czech Republic Faculty of Science, Department of Cell Biology, Charles University in Prague, Prague, Czech Republic Neurodegeneration Laboratory, Department of Anesthesiology, University of California, San Diego, La Jolla, CA, USA Sanford Consortium for Regenerative Medicine, San Diego, La Jolla, CA, USA. 3. Laboratory of Cell Regeneration and Plasticity, Institute of Animal Physiology and Genetics, v.v.i., AS CR, Libechov, Czech Republic. 4. Laboratory of Cell Regeneration and Plasticity, Institute of Animal Physiology and Genetics, v.v.i., AS CR, Libechov, Czech Republic Department of Neurology, Massachusetts General Hospital, Boston, MA, USA. 5. Vector Development Laboratory, Human Gene Therapy Program, Department of Pediatrics, University of California, San Diego, La Jolla, CA, USA. 6. Neurodegeneration Laboratory, Department of Anesthesiology, University of California, San Diego, La Jolla, CA, USA. 7. Neurodegeneration Laboratory, Department of Anesthesiology, University of California, San Diego, La Jolla, CA, USA Sanford Consortium for Regenerative Medicine, San Diego, La Jolla, CA, USA. 8. Novartis Institutes for Biomedical Research, Neuroscience Discovery, Basel, Switzerland IRBM Promidis, Pomezia, Italy. 9. Department of Genetics and Reproduction, Veterinary Research Institute, Brno, Czech Republic. 10. Department of Clinical Genetics and Fetal Medicine, Palacky University, University Hospital Olomouc, Olomouc, Czech Republic. 11. Department of Biological Chemistry University of California, Irvine, CA, USA Department of Psychiatry and Human Behavior, University of California, Irvine, CA, USA. 12. Department of Biological Chemistry University of California, Irvine, CA, USA Department of Psychiatry and Human Behavior, University of California, Irvine, CA, USA Department of Neurobiology and Behavior University of California, Irvine, CA, USA. 13. Institute of Information Theory and Automation v.v.i., AS CR, Prague, Czech Republic. 14. Laboratory for Study of Mitochondrial Disorders, First Faculty of Medicine, Department of Pediatrics and Adolescent Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic. 15. CHDI Foundation, Princeton, NY, USA. 16. Department of Pharmacological Sciences and Centre for Stem Cell Research, Università degli Studi di Milano, Milan, Italy. 17. Department of Neurology, Massachusetts General Hospital, Boston, MA, USA. 18. Neurodegeneration Laboratory, Department of Anesthesiology, University of California, San Diego, La Jolla, CA, USA Sanford Consortium for Regenerative Medicine, San Diego, La Jolla, CA, USA Institute of Neurobiology, Slovak Academy of Sciences, Kosice, Slovak Republic.
Abstract
BACKGROUND: Some promising treatments for Huntington's disease (HD) may require pre-clinical testing in large animals. Minipig is a suitable species because of its large gyrencephalic brain and long lifespan. OBJECTIVE: To generate HD transgenic (TgHD) minipigs encoding huntingtin (HTT)1-548 under the control of human HTT promoter. METHODS: Transgenesis was achieved by lentiviral infection of porcine embryos. PCR assessment of gene transfer, observations of behavior, and postmortem biochemical and immunohistochemical studies were conducted. RESULTS: One copy of the human HTT transgene encoding 124 glutamines integrated into chromosome 1 q24-q25 and successful germ line transmission occurred through successive generations (F0, F1, F2 and F3 generations). No developmental or gross motor deficits were noted up to 40 months of age. Mutant HTT mRNA and protein fragment were detected in brain and peripheral tissues. No aggregate formation in brain up to 16 months was seen by AGERA and filter retardation or by immunostaining. DARPP32 labeling in WT and TgHD minipig neostriatum was patchy. Analysis of 16 month old sibling pairs showed reduced intensity of DARPP32 immunoreactivity in neostriatal TgHD neurons compared to those of WT. Compared to WT, TgHD boars by one year had reduced fertility and fewer spermatozoa per ejaculate. In vitro analysis revealed a significant decline in the number of WT minipig oocytes penetrated by TgHD spermatozoa. CONCLUSIONS: The findings demonstrate successful establishment of a transgenic model of HD in minipig that should be valuable for testing long term safety of HD therapeutics. The emergence of HD-like phenotypes in the TgHD minipigs will require more study.
BACKGROUND: Some promising treatments for Huntington's disease (HD) may require pre-clinical testing in large animals. Minipig is a suitable species because of its large gyrencephalic brain and long lifespan. OBJECTIVE: To generate HD transgenic (TgHD) minipigs encoding huntingtin (HTT)1-548 under the control of humanHTT promoter. METHODS: Transgenesis was achieved by lentiviral infection of porcine embryos. PCR assessment of gene transfer, observations of behavior, and postmortem biochemical and immunohistochemical studies were conducted. RESULTS: One copy of the humanHTT transgene encoding 124 glutamines integrated into chromosome 1 q24-q25 and successful germ line transmission occurred through successive generations (F0, F1, F2 and F3 generations). No developmental or gross motor deficits were noted up to 40 months of age. Mutant HTT mRNA and protein fragment were detected in brain and peripheral tissues. No aggregate formation in brain up to 16 months was seen by AGERA and filter retardation or by immunostaining. DARPP32 labeling in WT and TgHD minipig neostriatum was patchy. Analysis of 16 month old sibling pairs showed reduced intensity of DARPP32 immunoreactivity in neostriatal TgHD neurons compared to those of WT. Compared to WT, TgHD boars by one year had reduced fertility and fewer spermatozoa per ejaculate. In vitro analysis revealed a significant decline in the number of WT minipig oocytes penetrated by TgHD spermatozoa. CONCLUSIONS: The findings demonstrate successful establishment of a transgenic model of HD in minipig that should be valuable for testing long term safety of HD therapeutics. The emergence of HD-like phenotypes in the TgHD minipigs will require more study.
Entities:
Keywords:
AGERA assay; DARPP32; FISH analysis; Huntington's disease; TR-FRET assay; immunohistochemistry; large animal model; lentiviral transgenesis; mRNA and protein expression; minipigs; mutant huntingtin; spermatozoa
Authors: Sean Moran; Tim Chi; Melinda S Prucha; Kwang Sung Ahn; Fawn Connor-Stroud; Sherrie Jean; Kenneth Gould; Anthony W S Chan Journal: Theriogenology Date: 2015-03-25 Impact factor: 2.740
Authors: Ting Lu; Yingchun Zheng; Haiying Yang; Buling Wu; Jun Xiong; Cheng Huang; Yuhua Pan; Meiyi Li; Fei He; Fu Xiong Journal: Nan Fang Yi Ke Da Xue Xue Bao Date: 2019-09-30