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

2. mechanisms of brain diseases neurodegenerative disorders epilepsy anxiety depression stress physiological and diseased brain functioning & development from genes to behavior therapy medicine basicapplied research pharmaceutical industry 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.

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

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

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

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

7. 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 Mission of the Medical Gene Technology Division Transgenic Unit Animal Technology Unit MGD IEMHAS

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

9. Virtual tour to MGD

10. Engine room airflow: 35.000 m 3 /h Regulated parameters: temperature humidity (60%) pressure Air filtering: trough multiple filters HEPA filters at SPF level Water: ultra filtered exchange rate: 12 X/h Ventilation: Food: autoclaved Light: regulated

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

12. Washing machine Bedding disposal station

13. SPF hallwayMD hallway

14. Mouse house

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

17. Research lab

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

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

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

21. Transgenic Technology Unit

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

23. 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)

24. transgenic models for brain diseases genetic modification of neuronal cell types, circuits and signaling 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 Transgenic technical developments and projects Group of Molecular Biology and Genetics projects

25. Microinjecting laboratory behind the barrier transfer window

27. DNA construct microinjection embryo transfer genotyping analysis Generating transgenic mouse models by pronuclear injection

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

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

30. 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

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

32. Conventional level trypsin treatment SPF level ♀ SPF foster mother checking hygienic status after weaning “clean” colony barrier ♂ ♀ X “dirty” colony 1 cell stage embryo collection superovulation Rederivation 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