Dobivanje i korištenje transgeničnih životinja Doc.dr.sc. Dinko Mitrečić, dr.med. Voditelj Laboratorija za matične stanice Hrvatski institut za istra živanje mozga Medicinski fakultet Sveučilišta u Zagrebu Geni - genske preinake… Značenje genskog zapisa (genoma)? Uloga gena? Kolika mi je sklonost da ću oboljeti od…? Koliko ću dugo živjeti? Tržište znanja! Kako otkriti ulogu gena? Klasična genetika FENOTIP ULOGA GENA ? PROMJENE FENOTIPA KOJI JE GEN ZAHVAĆEN? Laboratorijske životinje kao predmet proučavanja Zašto miševi? Male životinje, velika legla, kratko generacijsko razdoblje, dobro podnose život u zatočeništvu, troškovi niski Visoko srođene linije Niz generacija sparivanje životinja iz istog legla Sve životinje genetski iste (poput blizanaca) Dokaz: nema odbacivanja presatka tc/tc E12,5 Fotografije preuzete iz Colour Atlas of Congenital Malformation Syndromes Kako otkriti ulogu gena? Obrnuta genetika GEN MUTACIJA FENOTIP Kako odrediti ulogu gena? ili Koja genetska preinaka? Onemogućavanje uloge gena (loss of function, knock-out) Nadodavanje uloge gena (gain of function, transgenični miš) Transgenični miš: miš s dodatnom kopijom gena u genomu ili bilo koji genetski preinačeni miš The mouse gene Noto is expressed in the tail bud and essential for its morphogenesis Marica Zizic Mitrecic, Dinko Mitrecic, Roland Pochet, Ljiljana Kostovic-Knezevic, Srecko Gajovic Laboratory for Neurogenetics and Genetics of Development, Croatian Institute for Brain Research, School of Medicine, University of Zagreb, Zagreb, Croatia Department of Otorhinolaryngology, Head and Neck Surgery, University Hospital Center Zagreb, Zagreb, Croatia Laboratoire d'histologie générale, de neuroanatomie et de neuropathologie, Faculté de Médecine, Université Libre de Bruxelles, Bruxelles, Belgium Kako načiniti transgeničnog miša? Injiciranjem oocite (engl. Oocyte injection) Injicira se DNA koja sadrži dodatnu kopiju gena Takva DNA se konstruira postupcima bakterijske genetike (pomoću plazmida) Analyse the progeny by PCR or Southern blotting Transgene DNA Production of transgenic mice by pronuclear microinjection Pronuclear injection of DNA into fertilized ova Injection pipette containing DNA Holding pipette Fertilised ova (> 1 day old) Introduction of correctly modified stem cells in “host” blastocysts Injected blastocyst Host blastocyst Injection pipette Holding pipette Host’s stem cell mass injected stem cells Pokaži film! Eppendorf mikroinjektor P: Kako knock-out-irati gen? O: Homolognom rekombinacijom Ciljana promjena gena postiže se homolognom rekombinacijom Ciljana promjena gena postiže se homolognom rekombinacijom Ciljana promjena gena postiže se homolognom rekombinacijom Embrionalne matične stanice (engl. embryonic stem (ES) cells) Od kuda se dobivaju embrionalne matične stanice? Zametak na stadiju blastociste Embrionalne matične stanice (engl. embryonic stem (ES) cells) Early mouse development From Sedivy & Joyner “Gene Targeting” 1992 Razvoj mišjeg zametka Izolacija mišje maternice Ispiranje (flushing) mišje maternice – dobivanje blastocista Embryonic Stem Cell Isolation remove zona pellucida from blastocyst Microsurgery Immunosurgery Blastocyst Outgrowth antibody binding cut TE dissect off ICM trypsinize pipet off ICM complement TE cells lyse trypsinize plate on feeder cell layer Embrionalne matične stanice (engl. embryonic stem (ES) cells) Dokazi totipotentnosti embrionalnih matičnih stanica • In vitro (u kulturi stanica) • In vivo (vratiti ih natrag u razvitak zametka) Embryoid bodies Undifferentiated ES cells 4 days up to 9 days Culture in suspension + retinoic acid Plating on substrate Neuron-like cells after differentiation of ES cells (10-6 M retinoic acid, after 5 days of culture) 10-7 M retinoic acid after 2 days of culture Anti NF-M - fluorescein 10-7 M retinoic acid after 9 days Anti GFAP - rhodamine Pax6 expression (10-6 M retinoic acid, after 2 days of culture) Kamo se mogu ugraditi embrionalne matične stanice? opet u blastocistu Superovulation -3 – 5 wk old females (prepubescent age) - treatment with 5 i.u. of PMSG (pregnant mare’s serum gonadotropin) intraperitoneally, mimicking folliclestimulating hormone on day –2 (i.e. Saturday at 14 h) - treatment with 5 i.u. of hCG (human chorionic gonadotropin) 46 – 48 h upon PMSG treatment (i.e. Monday at 12 h) - mating set up withihn 6 h after the hCG treatment Od gentski preinačenih embrionalnih matičnih stanica do genetski preinačenog miša ES cell Chimera Production Blastocyst Injection Aggregation Flush embryos Uterus E3.5 Oviduct E2.5 Morula Blastocyst ES cells Remove zona pellucida Darning needle aggregation Recover 1-2 hours culture overnight implant into pseudopregnant surrogate mother Mating set up for transfer of 3.5 day embryo Donors of embryo Acceptors of embryo (pseudo-pregnant females, foster mothers) Fertile female X Fertile male Fertile female X Vasectomised (sterile) male Breeding start: Monday, Plug check: Tuesday Breeding start: Tuesday, Plug check: Wednesday Coat color difference! Coat Color Chimeras Chimeric Progeny Chimera ES cell injection into blastocyst C57BL/6 Blastocyst (black) agouti black ES cells 129/SvJ (agouti) Prijenos zametaka u lažno trudnu ženku -/wt -/wt wt/wt wt/wt wt/wt S.M. PCR -/wt wt/wt wt/wt no DNA no result 320bp 180bp Kako prepoznati u kojem klonu embrionalnih matičnih stanica je došlo do homologne rekombinacije? Embrionalne matične stanice koje su doživjele homolognu rekombinaciju prepoznate su hibridizacijom po Southernu Kako ću dokazati da sam zaista onemogućio gen koji proučavam? Koja je posljedica onemogućavanja gena? Kakav je fenotip homozigota? Koja je uloga proučavanog gena? The use of genetically modified mouse Gene function Mouse models for human disease Novel diagnostic procedures Novel therapeutic approaches Drug testing on mouse mutants Charcot, J., Joffroy, A: Deux cas d’atrophie musculaire progressive avec lesions de la substance grise et des faisceaux antero-lateraux de la moelle epiniere. Arch. Physiol. Neurol. Pathol. 2, 744–754. 1869. I am so tired and I simply can not play well anymore... …the bad news is this “lateral sclerosis”…they told me it is probably caused by some germ... Prevalence: 4 to 6 in 100,000 (motor neuron disease No.1) - 90% sporadic (SALS) (wide range of mutations without clear common factor) - 10% familial (FALS) SOD1 (superoxide dismutase 1) VAPB (vesicle associated membrane protein) SETX (senatexin) DCTN1 (dynectin) ALSIN (ALS2) TDP-43 SOD1 (Cu/Zn-SOD1) Rosen DR et al. Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature. 1993 Mar 4;362(6415):59-62 The SOD-catalysed dismutation of superoxide : M(n+1)+ − SOD + O2− → Mn+ − SOD + O2 Mn+ − SOD + O2− + 2H+ → M(n+1)+ − SOD + H2O2. 153 amino acids with 134 reported distinct amino acid changes. All of these mutations result in essentially the same disease, more or less rapidly progressive ALS. Almost any alteration in the structure of SOD1 gene will cause ALS and no other pathology. Some SOD1 mutations change tertiary conformation drastically, some not at all. Some change binding of Cu, some Zn, some both, some do not change anything. KO SOD1 mutants and overexpressing mutants showed that ALS pathology is independent of any kind of enzyme activity. Spontaneous SOD1 mouse mutant were reported as phenotypically normal (Luche et al., 1997). Basic prerequisite: multicopy presence of SOD1 (human, murine, mutated anyhow…) Problem of life lenghth? Genetics activity protein conformation … protein And what about mutSOD1 interactions ? - mutSOD1 proteins are more susceptible to form detergent-insoluble SOD1 oligomers that progressively aggregate and form inclusion bodies, which are the hallmark of ALS pathology. - generally, mutSOD1 is more sensitive to low levels of oxidative stress than wtSOD1 mitochondrial dysfunction protein aggregation oxidative chemistry synapse disactivity bad neigborhood structural and transport defects of axons 1. Cell replacement therapy Dobivanje i korištenje transgeničnih životinja Doc.dr.sc. Dinko Mitrečić, dr.med. Voditelj Laboratorija za matične stanice Hrvatski institut za istra živanje mozga Medicinski fakultet Sveučilišta u Zagrebu
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