Transgene verification and technical improvement in the establisment of transgenic miR-32 mouse line

The main challenge to be faced with prostate cancer (PCa) is the segregation and treatment of patients with life-threatening disease. As current prognostic, diagnostic and therapeutic tools are insufficient, solution is tried to be found from new biomarkers. One of these could be microRNA miR-32,...

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Main Author: Kakkonen, Maija
Other Authors: Matemaattis-luonnontieteellinen tiedekunta, Faculty of Sciences, Bio- ja ympäristötieteiden laitos, Department of Biological and Environmental Science, University of Jyväskylä, Jyväskylän yliopisto
Format: Master's thesis
Language:eng
Published: 2015
Subjects:
Online Access: https://jyx.jyu.fi/handle/123456789/47395
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author Kakkonen, Maija
author2 Matemaattis-luonnontieteellinen tiedekunta Faculty of Sciences Bio- ja ympäristötieteiden laitos Department of Biological and Environmental Science University of Jyväskylä Jyväskylän yliopisto
author_facet Kakkonen, Maija Matemaattis-luonnontieteellinen tiedekunta Faculty of Sciences Bio- ja ympäristötieteiden laitos Department of Biological and Environmental Science University of Jyväskylä Jyväskylän yliopisto Kakkonen, Maija Matemaattis-luonnontieteellinen tiedekunta Faculty of Sciences Bio- ja ympäristötieteiden laitos Department of Biological and Environmental Science University of Jyväskylä Jyväskylän yliopisto
author_sort Kakkonen, Maija
datasource_str_mv jyx
description The main challenge to be faced with prostate cancer (PCa) is the segregation and treatment of patients with life-threatening disease. As current prognostic, diagnostic and therapeutic tools are insufficient, solution is tried to be found from new biomarkers. One of these could be microRNA miR-32, which is possible up-regulated target of androgen receptor in aggressive PCa. Btg2 and Klf2 have further been suggested to be potential down-regulated miR-32 targets. To study hypothesized miR-32 oncogenicity in vivo, transgenic mice over-expressing miR-32 specifically in the prostate are established. Establishment of transgenic mouse line is a prolonged process, in which transgene functionality needs to be verified. Additionally, more than one so called founder line needs to be created from the same gene construct to confirm the phenotype. At the time of this study one line with verified miR-32 expression was established and more founder lines were screened for. In the first part of this study, transgene functionality i.e. expression and its effect on possible target genes was examined with quantitative PCR in the prostate samples of one new founder line. In the second part of this study, the suitability of PaxGene method for future studies was piloted. Molecular fixative PaxGene was used for the first time for prostate fixation in a desire to preserve both tissue morphology and integrity of nucleic acids from the same sample enabling one prostate to be used for both histopathological examination and molecular analyses. Traditionally these have been done from different prostates, as morphology preserving fixation methods have degraded RNA. Molecular analyses have further been done separately from different prostate lobes. In the one prostate method not only financial benefits but also ethical improvements would be achieved, as mouse individuals can be spared. Importantly, the transgene-phenotype conclusions would be more reliable too. To study PaxGenes feasibility, its RNA preservation ability was assessed by surveying the RNA integrity with BioAnalyzer. In addition, as the whole prostate cross-section with different lobes and adjacent tissues were now included in the RNA extraction as well, different tissue and prostate markers were used to study the possible effects of these issues on transgene detection. Clear transgene expression was detected only in two samples, whereas in rest of the samples the expression was of low level. Further, down-regulation of neither Btg2 nor Klf2 was observed in the transgenic samples. However, the RNA integrity PaxGene provided was on the same sufficient level that the traditional Trizol method constantly provides. Prostate marker expression profiles reveal that varying prostate lobe ratios may affect to transgene detection and be the reason for the low transgene expression values. However, prostate adjacent tissues most likely do not disturb the detection. It is also highly possible that the PaxGene procedure itself does not have an influence, but instead transgene is silenced. As a conclusion, PaxGene is a possible choice of method for future studies, if constant transgene detection can be verified even with varying prostate lobe ratios. PaxGene provides sufficient intact RNA and thus it could be possible used in in situ-hybridization based expression detection as well. In any case, not all the transgenic samples here have the desired transgene over-expression and thus it can not be verified that the transgene functions properly. For this reason, additional samples need to be examined in subsequent founder lines. Only with verified transgene over-expression reliable conclusions of the connections between the transgene and PCa can be made. In addition, possible down-regulation of Btg2 and Klf2 should be examined again in subsequent samples, before any final conclusions of their role as miR-32 targets are made. Suurin haaste eturauhassyöpään liittyen on erottaa ja hoitaa ne potilaat, joiden syöpä on henkeä uhkaava. Tämän hetkiset prognostiset, diagnostiset ja terapeuttiset menetelmät ovat riittämättömiä, minkä vuoksi ratkaisua yritetään etsiä uusista biomarkkereista. Yksi tällainen saattaa olla mikroRNA miR-32, joka mahdollisesti on androgeenireseptorin yli-ilmennetty kohdegeeni aggressiivisessa eturauhassyövässä. Tämän lisäksi Btg2 ja Klf2 ovat potentiaalisia ali-ilmennettyjä miR-32:n kohdegeenejä. miR-32:n mahdollista onkogeenisyyttä tutkitaan nyt in vivo transgeenisillä hiirillä, jotka yli-ilmentävät miR-32:ta spesifisesti eturauhasessa. Transgeenisen hiirilinjan luominen on pitkällinen prosessi, jossa transgeenin ilmeneminen tulee verifioida. Tämän lisäksi yhdestä transgeenikonstruktista tulee luoda useampi niin kutsuttu perustajalinja, jotta voidaan varmistua transgeenin fenotyypissä aiheuttamista muutoksista. Tämän tutkimuksen aikoihin transgeenin toimivuus oli varmistettu yhdestä perustajalinjasta ja seuraavia linjoja seulottiin. Tämän työn ensimmäisessä osassa tutkittiin transgeenin ilmentymistä ja sen vaikutusta mahdollisiin kohdegeeneihin kvantitatiivisen PCR:n avulla yhden uuden perustajalinjan hiirien eturauhasissa. Työn toisessa osassa testattiin PaxGene menetelmän soveltuvuutta jatkokäyttöä varten. Hiirten eturauhaset oli fiksattu PaxGene:n molekyylifiksatiivilla, joka lupaa säilöä sekä näytteen morfologian että nukleiinihapot tarkoittaen, että samaa eturauhasta voidaan käyttää sekä histopatologisiin että molekulaarisiin tutkimuksiin. Perinteisesti nämä tutkimukset on tehty eri eturauhasista, sillä morfologian säilöneet fiksaatiomenetelmät ovat hajottaneet RNA:n. Lisäksi molekulaariset tutkimukset on tehty jokaisesta eturauhaslohkosta erikseen. Yhden eturauhasen käyttäminen toisi taloudellisen edun lisäksi eettisiä etuja, kun käytettyjen hiiriyksilöiden määrä vähenisi. Tärkeä seikka on myös, että johtopäätökset transgeenin ja fenotyypin suhteesta olisivat luotettavampia kuin aiemmin. PaxGene:n käyttökelpoisuuden selvittämiseksi sen RNA:n säilömiskykyä arvioitiin tutkimalla RNA:n eheys BioAnalyzer:lla. Lisäksi eri kudos- ja eturauhasmarkkereiden avulla pyrittiin selvittämään vaikuttavatko eturauhasläpileikkeiden muut kudokset tai vaihtelevat eturauhaslohkokoostumukset transgeenin havainnointiin, sillä myös nämä olivat mukana RNA:n eristyksessä. Transgeenin selkeä ilmeneminen havaittiin vain kahdessa näytteessä, kun lopuissa näytteistä se oli hyvin matalaa. Transgeenisissä näytteissä ei myöskään havaittu Btg2:n tai Klf2:n alisäätelyä. Eheydeltään PaxGene näytteiden RNA oli kuitenkin samaa riittävää luokkaa kuin mitä Trizol jatkuvasti tarjoaa. Eturauhasmarkkereiden ekspressioprofiilit paljastavat, että vaihtelevat lohkokoostumukset saattavat vaikuttaa transgeenin havainnointiin ja olla syy matalille transgeenin ekspressioarvoille, mutta leikkeiden muut kudokset eivät kuitenkaan näyttäisi häiritsevän transgeenin detektiota. On kuitenkin myös hyvin mahdollista, ettei menettelytavalla ole vaikutusta havainnointiin, vaan transgeeni itsessään on hiljennetty. Johtopäätöksenä voidaan sanoa, että PaxGene on potentiaalinen menetelmä jatkoa ajatellen, mikäli transgeenin näkyminen pystytään varmentamaan, vaikka eri eturauhaslohkojen suhteelliset määrät vaihtelevat. PaxGene:llä saadaan joka tapauksessa riittävän eheää RNA:ta, minkä vuoksi kyseessä olevia näytteitä voisi mahdollisesti käyttää myös in situ-hybridisaatioon perustuvassa transgeenin havainnoinnissa. Loppujen lopuksi transgeenin ei voida kuitenkaan verifioida toimivan asianmukaisesti, sillä haluttu transgeenin yli-ilmeneminen ei näy kaikissa transgeenisissä näytteissä. Tämän vuoksi näytteitä on tutkittava lisää, sillä luotettavia johtopäätöksiä transgeenin ja eturauhassyövän yhteydestä voidaan tehdä ainoastaan transgeenin ollessa verifioitu. Lisäksi Btg2:n ja Klf2:n mahdollinen alisäätely tulisi tutkia uudelleen, ennen kuin lopullisia johtopäätöksiä näiden roolista miR-32:n kohdegeeneinä voidaan tehdä.
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fullrecord [{"key": "dc.contributor.advisor", "value": "Latonen, Leena", "language": "", "element": "contributor", "qualifier": "advisor", "schema": "dc"}, {"key": "dc.contributor.advisor", "value": "Yl\u00e4nne, Jari", "language": "", "element": "contributor", "qualifier": "advisor", "schema": "dc"}, {"key": "dc.contributor.author", "value": "Kakkonen, Maija", "language": null, "element": "contributor", "qualifier": "author", "schema": "dc"}, {"key": "dc.date.accessioned", "value": "2015-10-26T06:58:21Z", "language": "", "element": "date", "qualifier": "accessioned", "schema": "dc"}, {"key": "dc.date.available", "value": "2015-10-26T06:58:21Z", "language": "", "element": "date", "qualifier": "available", "schema": "dc"}, {"key": "dc.date.issued", "value": "2015", "language": null, "element": "date", "qualifier": "issued", "schema": "dc"}, {"key": "dc.identifier.other", "value": "oai:jykdok.linneanet.fi:1498445", "language": null, "element": "identifier", "qualifier": "other", "schema": "dc"}, {"key": "dc.identifier.uri", "value": "https://jyx.jyu.fi/handle/123456789/47395", "language": "", "element": "identifier", "qualifier": "uri", "schema": "dc"}, {"key": "dc.description.abstract", "value": "The main challenge to be faced with prostate cancer (PCa) is the segregation and treatment of\r\npatients with life-threatening disease. As current prognostic, diagnostic and therapeutic tools are insufficient,\r\nsolution is tried to be found from new biomarkers. One of these could be microRNA miR-32, which is\r\npossible up-regulated target of androgen receptor in aggressive PCa. Btg2 and Klf2 have further been\r\nsuggested to be potential down-regulated miR-32 targets. To study hypothesized miR-32 oncogenicity in\r\nvivo, transgenic mice over-expressing miR-32 specifically in the prostate are established. Establishment of\r\ntransgenic mouse line is a prolonged process, in which transgene functionality needs to be verified.\r\nAdditionally, more than one so called founder line needs to be created from the same gene construct to\r\nconfirm the phenotype. At the time of this study one line with verified miR-32 expression was established\r\nand more founder lines were screened for. In the first part of this study, transgene functionality i.e.\r\nexpression and its effect on possible target genes was examined with quantitative PCR in the prostate\r\nsamples of one new founder line. In the second part of this study, the suitability of PaxGene method for\r\nfuture studies was piloted. Molecular fixative PaxGene was used for the first time for prostate fixation in a\r\ndesire to preserve both tissue morphology and integrity of nucleic acids from the same sample enabling one\r\nprostate to be used for both histopathological examination and molecular analyses. Traditionally these have\r\nbeen done from different prostates, as morphology preserving fixation methods have degraded RNA.\r\nMolecular analyses have further been done separately from different prostate lobes. In the one prostate\r\nmethod not only financial benefits but also ethical improvements would be achieved, as mouse individuals\r\ncan be spared. Importantly, the transgene-phenotype conclusions would be more reliable too. To study\r\nPaxGenes feasibility, its RNA preservation ability was assessed by surveying the RNA integrity with\r\nBioAnalyzer. In addition, as the whole prostate cross-section with different lobes and adjacent tissues were\r\nnow included in the RNA extraction as well, different tissue and prostate markers were used to study the\r\npossible effects of these issues on transgene detection. Clear transgene expression was detected only in two\r\nsamples, whereas in rest of the samples the expression was of low level. Further, down-regulation of neither\r\nBtg2 nor Klf2 was observed in the transgenic samples. However, the RNA integrity PaxGene provided was\r\non the same sufficient level that the traditional Trizol method constantly provides. Prostate marker expression\r\nprofiles reveal that varying prostate lobe ratios may affect to transgene detection and be the reason for the\r\nlow transgene expression values. However, prostate adjacent tissues most likely do not disturb the detection.\r\nIt is also highly possible that the PaxGene procedure itself does not have an influence, but instead transgene\r\nis silenced. As a conclusion, PaxGene is a possible choice of method for future studies, if constant transgene\r\ndetection can be verified even with varying prostate lobe ratios. PaxGene provides sufficient intact RNA and\r\nthus it could be possible used in in situ-hybridization based expression detection as well. In any case, not all\r\nthe transgenic samples here have the desired transgene over-expression and thus it can not be verified that the\r\ntransgene functions properly. For this reason, additional samples need to be examined in subsequent founder\r\nlines. Only with verified transgene over-expression reliable conclusions of the connections between the\r\ntransgene and PCa can be made. In addition, possible down-regulation of Btg2 and Klf2 should be examined\r\nagain in subsequent samples, before any final conclusions of their role as miR-32 targets are made.", "language": "en", "element": "description", "qualifier": "abstract", "schema": "dc"}, {"key": "dc.description.abstract", "value": "Suurin haaste eturauhassy\u00f6p\u00e4\u00e4n liittyen on erottaa ja hoitaa ne potilaat, joiden sy\u00f6p\u00e4 on henke\u00e4\r\nuhkaava. T\u00e4m\u00e4n hetkiset prognostiset, diagnostiset ja terapeuttiset menetelm\u00e4t ovat riitt\u00e4m\u00e4tt\u00f6mi\u00e4, mink\u00e4\r\nvuoksi ratkaisua yritet\u00e4\u00e4n etsi\u00e4 uusista biomarkkereista. Yksi t\u00e4llainen saattaa olla mikroRNA miR-32, joka\r\nmahdollisesti on androgeenireseptorin yli-ilmennetty kohdegeeni aggressiivisessa eturauhassy\u00f6v\u00e4ss\u00e4. T\u00e4m\u00e4n\r\nlis\u00e4ksi Btg2 ja Klf2 ovat potentiaalisia ali-ilmennettyj\u00e4 miR-32:n kohdegeenej\u00e4. miR-32:n mahdollista\r\nonkogeenisyytt\u00e4 tutkitaan nyt in vivo transgeenisill\u00e4 hiirill\u00e4, jotka yli-ilment\u00e4v\u00e4t miR-32:ta spesifisesti\r\neturauhasessa. Transgeenisen hiirilinjan luominen on pitk\u00e4llinen prosessi, jossa transgeenin ilmeneminen\r\ntulee verifioida. T\u00e4m\u00e4n lis\u00e4ksi yhdest\u00e4 transgeenikonstruktista tulee luoda useampi niin kutsuttu\r\nperustajalinja, jotta voidaan varmistua transgeenin fenotyypiss\u00e4 aiheuttamista muutoksista. T\u00e4m\u00e4n\r\ntutkimuksen aikoihin transgeenin toimivuus oli varmistettu yhdest\u00e4 perustajalinjasta ja seuraavia linjoja\r\nseulottiin. T\u00e4m\u00e4n ty\u00f6n ensimm\u00e4isess\u00e4 osassa tutkittiin transgeenin ilmentymist\u00e4 ja sen vaikutusta\r\nmahdollisiin kohdegeeneihin kvantitatiivisen PCR:n avulla yhden uuden perustajalinjan hiirien eturauhasissa.\r\nTy\u00f6n toisessa osassa testattiin PaxGene menetelm\u00e4n soveltuvuutta jatkok\u00e4ytt\u00f6\u00e4 varten. Hiirten eturauhaset\r\noli fiksattu PaxGene:n molekyylifiksatiivilla, joka lupaa s\u00e4il\u00f6\u00e4 sek\u00e4 n\u00e4ytteen morfologian ett\u00e4 nukleiinihapot\r\ntarkoittaen, ett\u00e4 samaa eturauhasta voidaan k\u00e4ytt\u00e4\u00e4 sek\u00e4 histopatologisiin ett\u00e4 molekulaarisiin tutkimuksiin.\r\nPerinteisesti n\u00e4m\u00e4 tutkimukset on tehty eri eturauhasista, sill\u00e4 morfologian s\u00e4il\u00f6neet fiksaatiomenetelm\u00e4t\r\novat hajottaneet RNA:n. Lis\u00e4ksi molekulaariset tutkimukset on tehty jokaisesta eturauhaslohkosta erikseen.\r\nYhden eturauhasen k\u00e4ytt\u00e4minen toisi taloudellisen edun lis\u00e4ksi eettisi\u00e4 etuja, kun k\u00e4ytettyjen hiiriyksil\u00f6iden\r\nm\u00e4\u00e4r\u00e4 v\u00e4henisi. T\u00e4rke\u00e4 seikka on my\u00f6s, ett\u00e4 johtop\u00e4\u00e4t\u00f6kset transgeenin ja fenotyypin suhteesta olisivat\r\nluotettavampia kuin aiemmin. PaxGene:n k\u00e4ytt\u00f6kelpoisuuden selvitt\u00e4miseksi sen RNA:n s\u00e4il\u00f6miskyky\u00e4\r\narvioitiin tutkimalla RNA:n eheys BioAnalyzer:lla. Lis\u00e4ksi eri kudos- ja eturauhasmarkkereiden avulla\r\npyrittiin selvitt\u00e4m\u00e4\u00e4n vaikuttavatko eturauhasl\u00e4pileikkeiden muut kudokset tai vaihtelevat\r\neturauhaslohkokoostumukset transgeenin havainnointiin, sill\u00e4 my\u00f6s n\u00e4m\u00e4 olivat mukana RNA:n eristyksess\u00e4.\r\nTransgeenin selke\u00e4 ilmeneminen havaittiin vain kahdessa n\u00e4ytteess\u00e4, kun lopuissa n\u00e4ytteist\u00e4 se oli hyvin\r\nmatalaa. Transgeenisiss\u00e4 n\u00e4ytteiss\u00e4 ei my\u00f6sk\u00e4\u00e4n havaittu Btg2:n tai Klf2:n alis\u00e4\u00e4tely\u00e4. Eheydelt\u00e4\u00e4n PaxGene\r\nn\u00e4ytteiden RNA oli kuitenkin samaa riitt\u00e4v\u00e4\u00e4 luokkaa kuin mit\u00e4 Trizol jatkuvasti tarjoaa.\r\nEturauhasmarkkereiden ekspressioprofiilit paljastavat, ett\u00e4 vaihtelevat lohkokoostumukset saattavat vaikuttaa\r\ntransgeenin havainnointiin ja olla syy matalille transgeenin ekspressioarvoille, mutta leikkeiden muut\r\nkudokset eiv\u00e4t kuitenkaan n\u00e4ytt\u00e4isi h\u00e4iritsev\u00e4n transgeenin detektiota. On kuitenkin my\u00f6s hyvin mahdollista,\r\nettei menettelytavalla ole vaikutusta havainnointiin, vaan transgeeni itsess\u00e4\u00e4n on hiljennetty. Johtop\u00e4\u00e4t\u00f6ksen\u00e4\r\nvoidaan sanoa, ett\u00e4 PaxGene on potentiaalinen menetelm\u00e4 jatkoa ajatellen, mik\u00e4li transgeenin n\u00e4kyminen\r\npystyt\u00e4\u00e4n varmentamaan, vaikka eri eturauhaslohkojen suhteelliset m\u00e4\u00e4r\u00e4t vaihtelevat. PaxGene:ll\u00e4 saadaan\r\njoka tapauksessa riitt\u00e4v\u00e4n ehe\u00e4\u00e4 RNA:ta, mink\u00e4 vuoksi kyseess\u00e4 olevia n\u00e4ytteit\u00e4 voisi mahdollisesti k\u00e4ytt\u00e4\u00e4\r\nmy\u00f6s in situ-hybridisaatioon perustuvassa transgeenin havainnoinnissa. Loppujen lopuksi transgeenin ei\r\nvoida kuitenkaan verifioida toimivan asianmukaisesti, sill\u00e4 haluttu transgeenin yli-ilmeneminen ei n\u00e4y\r\nkaikissa transgeenisiss\u00e4 n\u00e4ytteiss\u00e4. T\u00e4m\u00e4n vuoksi n\u00e4ytteit\u00e4 on tutkittava lis\u00e4\u00e4, sill\u00e4 luotettavia johtop\u00e4\u00e4t\u00f6ksi\u00e4\r\ntransgeenin ja eturauhassy\u00f6v\u00e4n yhteydest\u00e4 voidaan tehd\u00e4 ainoastaan transgeenin ollessa verifioitu. Lis\u00e4ksi\r\nBtg2:n ja Klf2:n mahdollinen alis\u00e4\u00e4tely tulisi tutkia uudelleen, ennen kuin lopullisia johtop\u00e4\u00e4t\u00f6ksi\u00e4 n\u00e4iden\r\nroolista miR-32:n kohdegeenein\u00e4 voidaan tehd\u00e4.", "language": "fi", "element": "description", "qualifier": "abstract", "schema": "dc"}, {"key": "dc.description.provenance", "value": "Submitted using Plone Publishing form by Maija Kakkonen (anmakakk) on 2015-10-26 06:58:20.617183. Form: Pro gradu -lomake (https://kirjasto.jyu.fi/julkaisut/julkaisulomakkeet/pro-gradu-lomake). 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spellingShingle Kakkonen, Maija Transgene verification and technical improvement in the establisment of transgenic miR-32 mouse line PaxGene Solu- ja molekyylibiologia Cell and molecular biology 4013 eturauhassyöpä geenitekniikka mikro-RNA RNA markkerit
title Transgene verification and technical improvement in the establisment of transgenic miR-32 mouse line
title_full Transgene verification and technical improvement in the establisment of transgenic miR-32 mouse line
title_fullStr Transgene verification and technical improvement in the establisment of transgenic miR-32 mouse line Transgene verification and technical improvement in the establisment of transgenic miR-32 mouse line
title_full_unstemmed Transgene verification and technical improvement in the establisment of transgenic miR-32 mouse line Transgene verification and technical improvement in the establisment of transgenic miR-32 mouse line
title_short Transgene verification and technical improvement in the establisment of transgenic miR-32 mouse line
title_sort transgene verification and technical improvement in the establisment of transgenic mir 32 mouse line
title_txtP Transgene verification and technical improvement in the establisment of transgenic miR-32 mouse line
topic PaxGene Solu- ja molekyylibiologia Cell and molecular biology 4013 eturauhassyöpä geenitekniikka mikro-RNA RNA markkerit
topic_facet 4013 Cell and molecular biology PaxGene RNA Solu- ja molekyylibiologia eturauhassyöpä geenitekniikka markkerit mikro-RNA
url https://jyx.jyu.fi/handle/123456789/47395 http://www.urn.fi/URN:NBN:fi:jyu-201510263484
work_keys_str_mv AT kakkonenmaija transgeneverificationandtechnicalimprovementintheestablismentoftransgenicmir32mous