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
IEM-HAS is the only research institution in Hungary dedicated exclusively to medical research. Its activity is focused on basic biomedical research, primarily in the field of Neuroscience. 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
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 forms • • • for brain diseases joint major national research and development grants contract research and development initiated by industry contract research and development based on discoveries by the institute
GENE TECHNOLOGY AND DEVELOPMENTAL NEUROBIOLOGY head: G. SZABÓ CELLULAR AND NETWORK NEUROBIOLOGY ENDOCRINE NEUROBIOLOGY head: T. FREUND head: Z. LIPOSITS Medical Gene Technology Division animal & gene technology platform BEHAVIORAL NEUROBIOLOGY head: J. HALLER 12 research groups- ~70 scientists PHARMACOLOGY head: S. E. VIZI
The animal and gene technology platform Medical Gene Technology Division
Establishing the Medical Gene Technology Division at the Institute of Experimental Medicine Construction cost: 3. 5 million € sponsored by: IEMHAS building National granting agencies major equipments technology service activity running cost Medical Gene Technology Division MGD
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: Academic Research Institutions Pharmaceutical companies
Medical Gene Technology Division Animal Technology Unit 2. floor ~ 1000 SPF m 2 mouse MD Transgenic Technology Unit 2. microinjection szint embryo emelet-SPF embryo transfer DNA Virus Unit 1. floor Capacity: 20. 000 mice 4. 000 rats ground floor basement Genotyping and DNA Lab Receiving Animal Unit (quarantine)
Virtual tour to MGD
Ventilation: airflow: 35. 000 m 3/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 Engine room
autoclave behind the SPF barrier formalin chamber outside the SPF barrier personal air shower animal transfer window
Washing machine Bedding disposal station
SPF hallway MD hallway
Mouse house
Housing genetically modified mouse models in individually ventilated cages ventilating -unit ventilated-cage system
Research lab
Behavioral unit Complex behavioral testing of genetically modified mouse models • emotional tests • • aggression anxiety social avoidance motor tests learning and memory tests
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 transgenic colony management: 16 industry 2
Embryo freezing for long term storage National Mouse Embryo Bank superovulation with hormonal treatments ♀ X ♂ collection of one cell stage embryos trypsin treatment vitrification by Vajta OPS method number of frozen embryos >250/line number of frozen lines: 35 number of depopulated lines: 15
Transgenic Technology Unit
Medical Gene Technology Division Institute of Experimental Medicine Hungarian Academy of Sciences generating transgenic tools for genetic modification of well defined neuronal cell types transgenic models for neuro-psychiatric diseases anxiety epilepsy neurodegenerative diseases drug development
Transgenic service activities: in house and contract • Generation of transgenic models Transgene design and construction Generating transgenic mice by pronuclear injection Ø plasmid-based transgene Ø 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)
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
transfer window Microinjecting laboratory behind the barrier
Generating transgenic mouse models by pronuclear injection analysis DNA construct microinjection genotyping embryo transfer
Transgenic mouse models with genetically labeled neurons inhibitory GABAergic neurons hippocampus excitatory glutamatergic neurons cortex olfactory bulb cerebellar Purkinje cell pyramidal cells
Bacterial artificial chromosome-based transgenesis for more accurate genetic targeting: Parvalbumin/GFP-BAC transgenic mice bacterial artificial chromosome mouse parvalbumin gene ~180 kb GFP PV/GFP- modified BAC clone transgene isolation circular BAC Not. I digested BAC cerebellum 180 kb analysis PV-GFP BAC PV/GFP BAC fragment 190 kb 150 kb PV/GFP BAC 90 kb 45 kb 23 kb 10 kb 8 kb 6 kb 5 kb for targeting PV-containing GABAergic neurons hippocampus
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
Genotyping & DNA laboratory Genotyping: identifying heteroyzgous and homozygous transgenic mice by PCR Tests for more then 30 different genetic modifications Designing and constructing transgenes Transgenic colony: • establishment • maintenance • crossings Tail DNA extraction: 4154 PCR reaction: 9091 in close collaboration with Lab of Mol Biol & Gen
Rederivation SPF level Conventional level “clean” colony “dirty” colony ♀ X ♂ superovulation 1 cell stage embryo collection trypsin treatment barrier SPF foster ♀ mother checking hygienic status after weaning 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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