Biotechnology related activities at the Institute of Experimental Medicine, Hungarian Academy of

Sciences( IEM-HAS) Budapest, Hungary

Medical Gene Technology Division

GÁBOR SZABÓ MD, PHDszabog@koki.hu

May 3, 2007

www.koki.hu

mechanisms of brain diseases

• neurodegenerative disorders

• epilepsy• anxiety• depression• stress

physiological and diseased brain functioning & development from genes to behavior

physiological and diseased brain functioning & development from genes to behavior

therapymedicine

basic applied 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.

• 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

formsfor brain diseases

ENDOCRINE NEUROBIOLOGYhead: Z. LIPOSITS

ENDOCRINE NEUROBIOLOGYhead: Z. LIPOSITS

CELLULAR AND NETWORK NEUROBIOLOGY

head: T. FREUND

CELLULAR AND NETWORK NEUROBIOLOGY

head: T. FREUND

PHARMACOLOGYhead: S. E. VIZI

PHARMACOLOGYhead: S. E. VIZI

GENE TECHNOLOGY AND DEVELOPMENTAL NEUROBIOLOGY

head: G. SZABÓ

GENE TECHNOLOGY AND DEVELOPMENTAL NEUROBIOLOGY

head: G. SZABÓ

BEHAVIORAL NEUROBIOLOGY

head: J. HALLER

BEHAVIORAL NEUROBIOLOGY

head: J. HALLER

Medical Gene Technology Division

Medical Gene Technology Division

animal & gene technologyplatform

12 research groups- ~70 scientists

The animal and gene technology platform

Medical Gene Technology Division

IEMHAS

Medical Gene Technology Division

running cost

Establishing the Medical Gene Technology Division at the Institute of Experimental Medicine

technology

major equipments

major equipments

buildingbuilding

building

MGDMGD

sponsored by: National granting agencies

Construction cost: 3.5 million €

service activity

• providing animal and gene technology support for the research activity of the institute• providing transgenic technology serviceincluding breeding and generation of transgenicmodels for:

AcademicResearch Institutions

Pharmaceuticalcompanies

Mission of the Medical Gene Technology Division

Transgenic Unit

Animal Technology Unit

MGDMGD

IEMHAS

AnimalTechnology 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

DNAembryo

Medical Gene Technology Division

mouseembryo transfer

~ 1000 m2

Capacity: 20.000 mice 4.000 rats

Virtual tour to MGD

Engine room

airflow: 35.000 m3/h

Regulated parameters:• temperature• humidity (60%)• pressure

Air filtering:trough multiple filtersHEPA filters at SPF level

Water: ultra filtered

exchange rate: 12 X/h

Ventilation:

Food: autoclaved

Light: regulated

formalin chamber

outside the SPF barrier

behind the SPF barrier

autoclave

personal air shower

animal transfer window

Washing machine Bedding disposal station

SPF hallway MD hallway

Mouse house

ventilated-cage system

ventilating -unit

Housing genetically modified mouse models inindividually ventilated cages

Research lab Research lab

Behavioral unitComplex 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

academy industry

transgenic colony management: 16 2

• Import/export of mouse models

• Colony maintenance and breeding

• Timed embryo production

• Embryo freezing and storage

National Mouse Embryo Bank

Embryo freezing for long term storage

number of frozen lines: 35number of depopulated lines: 15

vitrificationby Vajta

OPS methodtrypsin

treatment

♂♀X

collection of one cell stage embryos

superovulation with hormonal treatments

number of frozen

embryos >250/line

Transgenic Technology Unit

anxietyepilepsy

drug development

generating transgenic tools for genetic modification of well defined neuronal cell

types

Medical Gene Technology DivisionInstitute of Experimental MedicineHungarian Academy of Sciences

transgenic models for neuro-psychiatric diseases transgenic models for neuro-psychiatric diseases

neurodegenerative diseases

Transgenic service activities:in house and contract

• Generation of transgenic modelsTransgene design and constructionGenerating transgenic mice by pronuclear injection

plasmid-based transgene BAC-based transgene

Establishing transgenic linesDetecting transgene expression at RNA and protein levels (level and distribution)

• Rederivation through embryo transfer

• Genotyping

• Somatic gene transfer (in utero electroporation)

transgenic models for brain diseases 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 developmentsand projects

Group of Molecular Biology and Genetics

• projects

Microinjecting laboratorybehind the barrier

transfer window

DNA construct microinjection

embryo transfer

genotyping

analysis

Generating transgenic mouse models by pronuclear injection

hippocampus

olfactory bulb

inhibitory GABAergicneurons

excitatory glutamatergicneurons

cortex

cerebellar Purkinje cell pyramidal cells

Transgenic mouse models with genetically labeled neurons

Bacterial artificial chromosome-based transgenesis for more accurate genetic targeting:Parvalbumin/GFP-BAC transgenic mice

PV/GFP BAC

cerebellum

for targeting PV-containingGABAergic neurons

190kb150kb

90kb

45kb23kb

10kb

8kb

6kb

5kb

PV/GFP BAC fragment

circular BAC

NotI digestedBAC

mouse parvalbumin gene~180 kb

GFP

PV/GFP- modified BAC clone

transgene isolation analysis

hippocampus

PV-GFP BAC

180 kb

bacterial artificial chromosome

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:identifying heteroyzgous and homozygous

transgenic mice by PCR

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: 4154PCR reaction: 9091

Tests for more then 30 different genetic modifications

Conventional level

trypsintreatment

SPF levelSPF level

♀ SPF foster mother

checking hygienic status after weaning

“clean” colony

barrier

♂♀X

“dirty” colony

1 cell stage embryocollection

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 progressindustry: 2