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Medical Gene Technology Division

Biotechnology related activities at the Institute of Experimental Medicine, Hungarian Academy of Sciences( IEM-HAS) Budapest, Hungary. www.koki.hu. Medical Gene Technology Division. GÁBOR SZABÓ MD, PHD szabog@koki.hu. May 3, 2007.

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Medical Gene Technology Division

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  1. Biotechnology related activities at the Institute of Experimental Medicine, Hungarian Academy of Sciences( IEM-HAS) Budapest, Hungary www.koki.hu Medical Gene Technology Division GÁBOR SZABÓ MD, PHD szabog@koki.hu May 3, 2007

  2. IEM-HASis the only research institution in Hungary dedicated exclusively tomedical research. Its activity is focused on basic biomedical research, primarily in the field ofNeuroscience. physiological and diseased brain functioning & development from genes to behavior basic applied research • mechanisms of brain diseases • neurodegenerative disorders • epilepsy • anxiety • depression • stress therapy medicine pharmaceutical industry

  3. Applied and translational research and development IEM-HAS Pharmaceutical industry Joint efforts goals • target identification, characterization and validation • drug efficacy tests • uncovering mechanisms of drug action for brain diseases forms • joint major national research and development grants • contract research and development initiated by industry • contract research and development based on discoveries by the institute

  4. GENE TECHNOLOGY AND DEVELOPMENTALNEUROBIOLOGY head:G. SZABÓ CELLULAR AND NETWORK NEUROBIOLOGY head: T. FREUND ENDOCRINE NEUROBIOLOGY head: Z. LIPOSITS Medical Gene Technology Division animal & gene technology platform • PHARMACOLOGY • head: S. E. VIZI BEHAVIORAL NEUROBIOLOGY head: J. HALLER 12 research groups- ~70 scientists

  5. The animal and gene technology platform Medical Gene Technology Division

  6. sponsored by: Establishing the Medical Gene Technology Division at the Institute of Experimental Medicine Construction cost: 3.5 million € National granting agencies IEMHAS major equipments building service activity technology running cost building Medical Gene Technology Division MGD

  7. Mission of the Medical Gene Technology Division IEMHAS Transgenic Unit Animal Technology Unit MGD • providing animal and gene technology support for the research activity of the institute • providing transgenic technology service including breeding and generation of transgenic models for: Pharmaceutical companies Academic Research Institutions

  8. Medical Gene Technology Division Transgenic Technology Unit Animal Technology Unit 2. floor 2. emelet-SPF szint embryo microinjection mouse SPF DNA embryo transfer ~ 1000 m2 MD Virus Unit 1. floor Capacity: 20.000 mice 4.000 rats ground floor Genotyping and DNA Lab Receiving Animal Unit (quarantine) basement

  9. Virtual tour to MGD

  10. Engine room Ventilation: airflow: 35.000 m3/h • Regulated parameters: • temperature • humidity (60%) • pressure Air filtering: trough multiple filters HEPA filters at SPF level exchange rate: 12 X/h Water:ultra filtered Food:autoclaved Light:regulated

  11. autoclave behind the SPF barrier formalin chamber outside the SPF barrier personal air shower animal transfer window

  12. Washing machine Bedding disposal station

  13. SPF hallway MD hallway

  14. Mouse house

  15. Housing genetically modified mouse models in individually ventilated cages ventilating -unit ventilated-cage system

  16. Research lab Research lab

  17. Behavioral unit Complex behavioral testing of genetically modified mouse models • emotional tests • aggression • anxiety • social avoidance • motor tests • learning and memory tests

  18. Animal technology service activities:in house and contract • Import/export of mouse models • Colony maintenance and breeding • Timed embryo production • Embryo freezing and storage academy industry transgenic colony management: 162

  19. number of frozen embryos >250/line Embryo freezing for long term storage National Mouse Embryo Bank superovulation with hormonal treatments ♂ ♀ X collection of one cell stage embryos vitrification by Vajta OPS method trypsin treatment number of frozen lines: 35 number of depopulated lines: 15

  20. Transgenic Technology Unit

  21. Medical Gene Technology Division Institute of Experimental Medicine Hungarian Academy of Sciences generating transgenictools for genetic modification of well defined neuronal cell types transgenic models for neuro-psychiatric diseases anxiety epilepsy neurodegenerative diseases drug development

  22. Transgenic service activities:in house and contract • Generation of transgenic models Transgene design and construction Generating transgenic mice by pronuclear injection • plasmid-based transgene • BAC-based transgene Establishing transgenic lines Detecting transgene expression at RNA and protein levels (level and distribution) • Rederivation through embryo transfer • Genotyping • Somatic gene transfer (in utero electroporation)

  23. Transgenic technical developments and projects • technical developments • BAC technology - developed • Tet inducible technology- in progress • „Cre-stop” technology- in progress • speedy mouse” technology - future plans • transgenic rat- future plans • “knockout” technology- future plans • projects genetic modification of neuronal cell types, circuits and signaling transgenic models for brain diseases Group of Molecular Biology and Genetics

  24. transfer window Microinjecting laboratory behind the barrier

  25. Generating transgenic mouse models by pronuclear injection analysis DNAconstruct microinjection genotyping embryo transfer

  26. Transgenic mouse models with genetically labeled neurons inhibitory GABAergic neurons excitatory glutamatergic neurons hippocampus cortex olfactory bulb cerebellar Purkinje cell pyramidal cells

  27. bacterial artificial chromosome mouse parvalbumin gene ~180 kb GFP PV/GFP- modified BAC clone cerebellum transgene isolation analysis 180 kb PV-GFP BAC PV/GFP BAC fragment circular BAC PV/GFP BAC 190kb 150kb 90kb 45kb 23kb 10kb 8kb 6kb 5kb NotI digested BAC for targeting PV-containing GABAergic neurons hippocampus Bacterial artificial chromosome-based transgenesis for more accurate genetic targeting: Parvalbumin/GFP-BAC transgenic mice

  28. Generated transgenic models* • GFP labeled neuronal cell-types : 7 (6 plasmid and 1 BAC constructs) • Genetically modified neuronal signaling: 4(plasmid constructs) • Transgenic model for neurodegenerative and other disorders: 5(4 plasmid and 1 BAC constructs) *12- inhouse 3- industry 1- academy

  29. Genotyping & DNA laboratory • Transgenic colony: • establishment • maintenance • crossings Genotyping: identifying heteroyzgous and homozygous transgenic mice by PCR Tests for more then 30 different genetic modifications Tail DNA extraction: 4154 PCR reaction: 9091 in close collaboration with Lab of Mol Biol & Gen Designing and constructing transgenes

  30. Rederivation SPF level Conventional level “clean” colony “dirty” colony ♂ ♀ X superovulation 1 cell stage embryo collection SPF foster mother ♀ barrier checking hygienic status after weaning trypsin treatment 61 lines were transferred to SPF facility through rederivation(42 lines from old facility and 12 imported lines). 11 rederivations are in progress and 5 are planned. academy: 6+ 9 in progress industry: 2

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