京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19047号 / 農博第2125号 / 新制||農||1032(附属図書館) / 学位論文||H27||N4929(農学部図書室) / 31998 / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 小川 順, 教授 喜多 恵子, 教授 栗原 達夫 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

La recombinaison homologue est un mécanisme de réparation de l’ADN extrêmement contrôlé et particulièrement chez les eucaryotes supérieurs. Dans les cellules méiotiques de ces derniers, où les cassures doubles brin de l’ADN sont programmées, les voies de crossing-over de la recombinaison homologue, qui génèrent de nouvelles combinaisons de gènes, sont restreintes. Dans les cellules somatiques, la recombinaison illégitime, qui assure majoritairement la réparation des cassures double brin de l’ADN, limite l’intégration ciblée du transgène par recombinaison homologue. Les entreprises de biotechnologie convoitent de maitriser la recombinaison homologue afin de contrôler d’une part le brassage génomique qui a lieu pendant la méiose, et d’autre part l’intégration du transgène dans le génome. Cette étude a porté sur le développement d’outils afin d’atteindre ces deux objectifs. Afin d’augmenter le brassage du génome, ayant lieu pendant la méiose, une version du promoteur OsDmc1b, active dans les cellules méiotiques, a été caractérisée chez le maïs. Des plantes sur-exprimant le gène ZmSpo11.1, sous contrôle de ce promoteur, ont ainsi été développées afin d’obtenir des lignées potentiellement hyper-recombinantes. Si la surexpression de ZmSpo11.1 permet effectivement d’augmenter le taux de crossing-over, il pourra être utilisé par les sélectionneurs afin d’accélérer l’introgression d’allèles d’intérêt dans des variétés élites. Concernant la mise en place d’une technique de ciblage de gène, deux stratégies, reposant sur l’utilisation de la méganucléase I-SceI, ont été testées. La démarche a nécessité trois éléments : un locus cible contenant le site de coupure I-SceI, une matrice de réparation et la séquence codant I-SceI (ou I-SceI::GR). La première stratégie, consistant à retransformer les lignées présentant le locus cible avec la matrice de réparation et I-SceI, ne semble pas exploitable car aucun évènement de ciblage de gène n’a été mis en évidence. La seconde stratégie, reposant sur l’assemblage des trois éléments par croisement, est beaucoup plus prometteuse. Malgré la faible activité d’I-SceI::GR, des évènements de recombinaison homologue ont été observés dans les tissus foliaires de certaines plantes. Du cal embryogène, développé à partir de ces dernières, a permis de régénérer des plantes présentant des évènements de ciblage de gène. Ces travaux ouvrent de nouvelles perspectives dans l’élaboration contrôlée d’OGM. / Homologous recombination is a DNA repair mechanism highly regulated in higher eukaryotes. In their meiotic cells, where DNA double-stranded breaks are programmed, the crossing-over pathway of homologous recombination, which generates new gene combinations, is limited in activity and genomic distribution. In somatic cells, illegitimate recombination, which mainly ensures DNA double-strand repair, limits the targeted integration of transgenes by homologous recombination. Biotechnology companies aim to master homologous recombination to control on the one hand the genomic mixing that occurs during meiosis, and on other hand, the integration of transgenes into the genome. This study focuses on the development of tools to achieve these two objectives.To increase genome mixing occurring during meiosis, a version of the OsDmc1b promoter active in maize meiotic cells was isolated. Then, plants over-expressing the ZmSpo11.1 gene under control of this promoter have been developed to obtain potentially hyper-recombinant lines. If ZmSPO11.1 overexpression increases the crossing over rate, it can be used by breeders to accelerate the introgression of alleles of interest into elite varieties. For the establishment of a gene targeting technique, two strategies based on the use of the I-SceI meganuclease were tested. These approaches involved the use of three elements which are: a target locus containing the cleavage site of I-SceI, a repair template and the sequence encoding I-SceI (or ISceI::GR). The first strategy, consisting of the retransformation of target locus lines with the repair template and I-SceI, does not seem workable because no gene targeting events were isolated. The second strategy, based on the assembly of the three components by crossing, is more promising. Despite the low activity of I-SceI::GR, homologous recombination events were observed in leaf tissues of certain plants. Embryogenic callus, developed from these plants, permitted the regeneration of plants with gene targeting events. This work opens new perspectives in the development of controlled GMO production.

<p>In the extracellular space, many specialized proteins are located to support cells and to mediate cell-cell signalling. One class of such molecules is heparan sulfate (HS) proteoglycans, which are proteins with different properties and locations but all of them decorated with long unbranched HS polysaccharide chains. During biosynthesis the HS chains are modified by sulfation and a C5-epimerase converts some glucuronic acid residues to iduronic acid. The patterns of the modifications vary distinctly between tissues and developing stages and give HS chains different affinity for biologically important proteins. Thus, the regulation of HS biosynthesis is likely to influence a wide variety of biological events.</p><p>This thesis focuses on the biosynthesis of HS in animals with targeted disruptions in genes important for HS production. The N-deacetylase N-sulfotransferase (NDST) is a key enzyme in HS biosynthesis, directing other modifications. We show that NDST isoforms have very different roles in HS biosynthesis. Inactivation of NDST1 affects HS biosynthesis in all tissues. In embryonic liver HS from NDST1-/- mice the N-sulfation was decresed with twothirds, while the absence of NDST2 did not affect HS structure. In the absence of NDST1 in the liver, however, NDST2 is active in HS N-sulfation. </p><p>In a study of embryonic stem cells lacking both NDST1 and NDST2, no N-sulfate groups could be detected. 6-O-sulfate groups were, however, still present at half of its normal level. This was an unexpected finding since 6-O-sulfotransferases have been thought to be strictly dependent on N-sulfate groups for substrate recognition.</p><p>By adapting an automated method for HS analysis to mammalian tissues, we could extend our analyses to more tissues and other transgene models. We also developed a protocol to create a sensitive “fingerprint” of HS structure. With these methods we could determine the individual HS structure of different mouse tissues. </p>

<p>Heparan sulfate (HS) is a ubiquitous polysaccharide on the cell surface and in the extracellular matrix. HS is an important actor in the regulation of cell signaling, especially in the developing embryo. In combination with cell culture and biochemical experiments, <i>in vivo</i> studies of genetically modified animals have pointed out the sulfation pattern of HS as highly important for binding of ligands, their receptors and other signaling modulators.</p><p>The sulfation pattern of an HS chain is gained by several modifying steps, performed by multiple enzymes during biosynthesis in the Golgi apparatus. By alterations of sulfation pattern, and the amount of sulfate groups, a cell can regulate the binding properties of its HS to different molecules. The most highly sulfated form of HS is called heparin, and can only be found intracellularly in mast cells.</p><p>This thesis describes the phenotypes and the alterations in HS/heparin biosynthesis of two genetically modified mouse strains deficient in N-deacetylase/N-sulfotransferase-1 (NDST1) and -2 (NDST2) respectively. We have found NDST1 to be important for correct sulfation of HS and that NDST2 is crucial in heparin biosynthesis. NDST2 deficient mice completely lack heparin and therefore have a severe mast cell phenotype. NDST1 deficient mice produce undersulfated HS and show several developmental disturbances. Some NDST1 embryos die in utero while the rest die neonatally due to breathing difficulties. Defect brain, eye and skeletal development has also been observed while some organs, such as the liver, appear to be largely unaffected. Several phenotypes are similar to defects seen in other mouse strains with impaired fibroblast growth factor and bone morphogenetic protein signaling, among others. This suggests the phenotypes of NDST1 deficient embryos to be of a multi factorial origin, in complete accordance to the many signaling pathways HS is suggested to modulate.</p>

<p>Heparan sulfate (HS) proteoglycans consist of sulfated HS chains covalently bound to core proteins. They are ubiquitously expressed, on the cell surface and in the extracellular matrix, throughout the body. During biosynthesis the HS chain is modified to generate a highly variable pattern of sulfated residues, able to interact with a wide variety of ligands, such as growth factors, morphogens and extracellular matrix molecules. The presence of HS proteoglycans is crucial during various developmental processes as they are involved in generation of morphogen gradients and influence the function of several growth factor pathways essential for tissue assembly and differentiation.</p><p>In this thesis the phenotypes of two mouse strains, deficient in different isoforms of the HS biosynthetic enzyme N-deacetylase/N-sulfotransferase (NDST) have been analyzed. In addition, NDST deficient embryonic stem (ES) cells have been analyzed with regard to HS structure and differentiation capacity. Mice deficient in NDST1 die peri-natally. The embryos display an overall low-sulfated HS and several developmental defects, with a lung phenotype as the predominant cause of death. Mice deficient in NDST2 lack sulfated heparin in connective tissue type mast cells while HS structure is unaltered. These results indicate that NDST1 is the isoform mainly responsible for HS biosynthesis during development. However, NDST1/2 deficient embryos do not survive beyond E5.5 and have a greatly disturbed morphology, suggesting that NDST2 has an essential role during early embryonic development. HS synthesized by NDST1/2 deficient ES cells had a total lack of N-sulfate groups while, interestingly, about half of the 6-O-sulfate groups remained. This result was unexpected since 6-O-sulfotransferases have been thought to be strictly dependent on N-sulfate groups for substrate recognition. Further characterization of the NDST1/2 deficient ES cells during in vitro differentiation demonstrated that the expression pattern of markers for all three germ layers was disturbed. In addition, it was demonstrated that NDST1 is not needed for mast cell development, that lack of NDST2 results in abnormal mast cells and that no mast cells is formed from NDST1/2 deficient ES cells.</p>

In the extracellular space, many specialized proteins are located to support cells and to mediate cell-cell signalling. One class of such molecules is heparan sulfate (HS) proteoglycans, which are proteins with different properties and locations but all of them decorated with long unbranched HS polysaccharide chains. During biosynthesis the HS chains are modified by sulfation and a C5-epimerase converts some glucuronic acid residues to iduronic acid. The patterns of the modifications vary distinctly between tissues and developing stages and give HS chains different affinity for biologically important proteins. Thus, the regulation of HS biosynthesis is likely to influence a wide variety of biological events. This thesis focuses on the biosynthesis of HS in animals with targeted disruptions in genes important for HS production. The N-deacetylase N-sulfotransferase (NDST) is a key enzyme in HS biosynthesis, directing other modifications. We show that NDST isoforms have very different roles in HS biosynthesis. Inactivation of NDST1 affects HS biosynthesis in all tissues. In embryonic liver HS from NDST1-/- mice the N-sulfation was decresed with twothirds, while the absence of NDST2 did not affect HS structure. In the absence of NDST1 in the liver, however, NDST2 is active in HS N-sulfation. In a study of embryonic stem cells lacking both NDST1 and NDST2, no N-sulfate groups could be detected. 6-O-sulfate groups were, however, still present at half of its normal level. This was an unexpected finding since 6-O-sulfotransferases have been thought to be strictly dependent on N-sulfate groups for substrate recognition. By adapting an automated method for HS analysis to mammalian tissues, we could extend our analyses to more tissues and other transgene models. We also developed a protocol to create a sensitive “fingerprint” of HS structure. With these methods we could determine the individual HS structure of different mouse tissues.

Heparan sulfate (HS) is a ubiquitous polysaccharide on the cell surface and in the extracellular matrix. HS is an important actor in the regulation of cell signaling, especially in the developing embryo. In combination with cell culture and biochemical experiments, in vivo studies of genetically modified animals have pointed out the sulfation pattern of HS as highly important for binding of ligands, their receptors and other signaling modulators. The sulfation pattern of an HS chain is gained by several modifying steps, performed by multiple enzymes during biosynthesis in the Golgi apparatus. By alterations of sulfation pattern, and the amount of sulfate groups, a cell can regulate the binding properties of its HS to different molecules. The most highly sulfated form of HS is called heparin, and can only be found intracellularly in mast cells. This thesis describes the phenotypes and the alterations in HS/heparin biosynthesis of two genetically modified mouse strains deficient in N-deacetylase/N-sulfotransferase-1 (NDST1) and -2 (NDST2) respectively. We have found NDST1 to be important for correct sulfation of HS and that NDST2 is crucial in heparin biosynthesis. NDST2 deficient mice completely lack heparin and therefore have a severe mast cell phenotype. NDST1 deficient mice produce undersulfated HS and show several developmental disturbances. Some NDST1 embryos die in utero while the rest die neonatally due to breathing difficulties. Defect brain, eye and skeletal development has also been observed while some organs, such as the liver, appear to be largely unaffected. Several phenotypes are similar to defects seen in other mouse strains with impaired fibroblast growth factor and bone morphogenetic protein signaling, among others. This suggests the phenotypes of NDST1 deficient embryos to be of a multi factorial origin, in complete accordance to the many signaling pathways HS is suggested to modulate.

Heparan sulfate (HS) proteoglycans consist of sulfated HS chains covalently bound to core proteins. They are ubiquitously expressed, on the cell surface and in the extracellular matrix, throughout the body. During biosynthesis the HS chain is modified to generate a highly variable pattern of sulfated residues, able to interact with a wide variety of ligands, such as growth factors, morphogens and extracellular matrix molecules. The presence of HS proteoglycans is crucial during various developmental processes as they are involved in generation of morphogen gradients and influence the function of several growth factor pathways essential for tissue assembly and differentiation. In this thesis the phenotypes of two mouse strains, deficient in different isoforms of the HS biosynthetic enzyme N-deacetylase/N-sulfotransferase (NDST) have been analyzed. In addition, NDST deficient embryonic stem (ES) cells have been analyzed with regard to HS structure and differentiation capacity. Mice deficient in NDST1 die peri-natally. The embryos display an overall low-sulfated HS and several developmental defects, with a lung phenotype as the predominant cause of death. Mice deficient in NDST2 lack sulfated heparin in connective tissue type mast cells while HS structure is unaltered. These results indicate that NDST1 is the isoform mainly responsible for HS biosynthesis during development. However, NDST1/2 deficient embryos do not survive beyond E5.5 and have a greatly disturbed morphology, suggesting that NDST2 has an essential role during early embryonic development. HS synthesized by NDST1/2 deficient ES cells had a total lack of N-sulfate groups while, interestingly, about half of the 6-O-sulfate groups remained. This result was unexpected since 6-O-sulfotransferases have been thought to be strictly dependent on N-sulfate groups for substrate recognition. Further characterization of the NDST1/2 deficient ES cells during in vitro differentiation demonstrated that the expression pattern of markers for all three germ layers was disturbed. In addition, it was demonstrated that NDST1 is not needed for mast cell development, that lack of NDST2 results in abnormal mast cells and that no mast cells is formed from NDST1/2 deficient ES cells.

Η RNase P είναι το ένζυμο που ωριμάζει το 5΄ άκρο των πρόδρομων μορίων tRNA, ενώ έχει βρεθεί και στις τρεις φυλογενετικές περιοχές, καθώς και σε υποκυτταρικά οργανίδια. Είναι ριβονουκλεοπρωτεϊνικής φύσεως στις περισσότερες περιπτώσεις, ενώ έχουν βρεθεί και ένζυμα RNase P αποκλειστικά πρωτεϊνικής φύσεως. Η υπομονάδα RNA των βακτηριακών ολοενζύμων είναι καταλυτικά ενεργή απουσία πρωτεϊνικών παραγόντων in vitro, καθιστώντας την ένα πραγματικό ριβοένζυμο. Η ικανότητα της RNase P να αναγνωρίζει συγκεκριμένες δομές στα μόρια των υποστρωμάτων της και όχι αλληλουχίες, δημιούργησε τη δυνατότητα χρήσης αυτού του ενζύμου ως ενός μοριακού εργαλείου για τη στόχευση πολλών ιικών και παθολογικών μορίων RNA in vitro και in vivo, καταστέλλοντας την έκφραση των μορίων αυτών, μέσω της τεχνολογίας των μορίων EGS (external guide sequence) και των ριβοενζύμων M1GS. Η RNase P, σύμφωνα με πολλές μελέτες, έχει δειχθεί ότι αποτελεί στόχο πολλών φαρμακευτικών παραγόντων, συμπεριλαμβανομένων πολλών γνωστών αντιβιοτικών, οι οποίοι κατά κύριο λόγο αναστέλλουν τη δραστικότητα του ενζύμου. Πρόσφατα δείχτηκε, μέσω αναλυτικής κινητικής μελέτης, ότι ένα μακρολίδιο, η σπιραμυκίνη, ενεργοποιεί σημαντικά τη δραστικότητα της βακτηριακής RNase P και του M1 RNA κατά ένα δοσοεξαρτώμενο τρόπο, λειτουργώντας έτσι ως μη-ειδικός ενεργοποιητής μικτού τύπου. Μέχρι σήμερα, στη διεθνή βιβλιογραφία, δεν έχει αναφερθεί άλλη ουσία η οποία προκαλεί θετική επίδραση στη δραστικότητα της RNase P. Στην παρούσα μελέτη, αρχικά μελετήθηκε η ενεργοποίηση της δραστικότητας της βακτηριακής RNase P και αποκαλύφθηκε ότι η σπιραμυκίνη δεν αλληλεπιδρά με ιοντικούς δεσμούς με το μόριο Μ1 RNA, αλλά προκαλεί αλλαγή διαμόρφωσης στο δομικό στοιχείο P10/11 του ριβοενζύμου. Το δομικό αυτό στοιχείο εμπλέκεται στην αναγνώριση του υποστρώματος, αποτέλεσμα το οποίο έρχεται σε συμφωνία με τις τιμές KD που προσδιορίστηκαν για το σύμπλοκο ριβοενζύμου–υποστρώματος, απουσία και παρουσία σπιραμυκίνης. Με δεδομένο ότι η σπιραμυκίνη δεν επηρεάζει την πρωτεϊνοσύνθεση ή τη δραστικότητα της RNase P των ευκαρυωτικών κυττάρων, κατασκευάστηκε ένα ριβοένζυμο M1GS, ώστε να ελεγχθεί η επίδραση του αντιβιοτικού στη δραστικότητα αυτού του ριβοενζύμου in vivo, σε καλλιεργούμενα ανθρώπινα κύτταρα ΗΕΚ293. Ως στόχος του συγκεκριμένου M1GS, επιλέχτηκε ο μεταγραφικός παράγοντας Ets2 λόγω της μεγάλης κλινικής σημασίας του, εφόσον έχει συσχετιστεί με αρκετούς τύπους καρκίνου και παθολογικές καταστάσεις, καθώς και με διαδικασίες διαφοροποίησης. Ο σπουδαίος ρόλος του Ets2, σε συνδυασμό με τα ελλιπή δεδομένα σχετικά με την έκφρασή του, είχαν αποτρέψει μέχρι σήμερα την αποτελεσματική στόχευσή του με τη χρήση των υπαρχουσών μεθοδολογιών που βασίζονται στο RNA, όπως το RNAi. Μετά από ανάλυση της δευτεροταγούς δομής του Ets2 mRNA, σχεδιάστηκαν δύο οδηγοί αλληλουχίες. Οι αλληλουχίες αυτές, αρχικά, δοκιμάστηκαν ως εξωτερικές οδηγοί αλληλουχίες (EGS) σε συνδυασμό με το βακτηριακό ολοένζυμο της RNase P. Η EGS303 (το νούμερο υποδεικνύει το νουκλεοτιδικό κατάλοιπο του στόχου που δρα η RNase P), εμφάνισε τη μεγαλύτερη ικανότητα να επάγει τη δράση της RNase P in vitro. Η οδηγός αυτή αλληλουχία, στη συνέχεια κλωνοποιήθηκε στο 3΄ άκρο του M1 RNA, παράγοντας το ριβοένζυμο M1GS303, το οποίο είναι δραστικό έναντι του μορίου–στόχου του in vitro. Η δραστικότητα του συγκεκριμένου ριβοενζύμου ενεργοποιείται εντυπωσιακά κατά 160% παρουσία σπιραμυκίνης. Προκειμένου να ελεγχθεί η δραστικότητα αυτού του ριβοενζύμου in vivo, το μόριο–στόχος και το ριβοένζυμο εκφράστηκαν ελεγχόμενα σε κύτταρα E. coli, προκαλώντας μείωση της έκφρασης του μορίου–στόχου από το M1GS303 κατά 95% μετά από 12 ώρες έκφρασης των μορίων. Μείωση στα ίδια επίπεδα ανιχνεύτηκε μόλις μετά από 4 ώρες έκφρασης εφόσον στα κύτταρα είχε προστεθεί σπιραμυκίνη, γεγονός που υποστηρίζει την εντυπωσιακά θετική επίδραση της σπιραμυκίνης επί της δραστικότητας του ριβοενζύμου. Η ίδια σειρά πειραμάτων επαναλήφθηκε σε ευκαρυωτικά κύτταρα, με έκφραση του ριβοενζύμου σε HEK293 κύτταρα. Η δραστικότητα του ριβοενζύμου προσδιορίστηκε ποιοτικά και ποσοτικά, από την έκφραση της χιμαιρικής φθορίζουσας πρωτεΐνης Ets2–EGFP (μόριο–στόχος), σε διαφορετικούς χρόνους έκφρασης. Παρατηρήθηκε ότι το M1GS δρα αποτελεσματικά έναντι του μορίου–στόχου του και σε ευκαρυωτικά κύτταρα in vivo, προκαλώντας μείωση στην έκφραση του Ets2, η οποία αυξάνεται επιπλέον παρουσία σπιραμυκίνης. Τα παραπάνω αποτελέσματα δείχνουν τη σημαντική ενεργοποίηση της δραστικότητας του M1GS σε ανθρώπινα κύτταρα και καθιστούν τη σπιραμυκίνη ένα σημαντικό ενεργοποιητή στη χρήση των ριβοενζύμων M1GS ως εργαλεία γονιδιακής αποσιώπησης. Ο συνδυασμός βελτιωμένων ριβοενζύμων M1GS με την παρουσία σπιραμυκίνης αυξάνει ακόμα περισσότερο την πρακτική χρήση της συγκεκριμένης τεχνολογίας τόσο in vitro όσο και in vivo, επιτυγχάνοντας ακόμα πιο αποτελεσματική αποσιώπηση της γονιδιακής έκφρασης. / RNase P is the enzyme that endonucleolytically cleaves the precursor tRNA transcripts to produce their mature 5΄ ends. It has been found in all three phylogenetic domains of life, as well as in subcellular organelles. In most cases, it has been described as a ribonucleoprotein complex. However, few RNase P enzymes that are exclusively proteinaceous have been also reported recentrly. The RNA subunit of bacterial holoenzyme is catalytically active in the absence of protein factors in vitro, making it a true ribozyme. The ability of RNase P to recognize specific structures in its substrate molecules instead of specific sequences, allowed the use of this enzyme as a molecular tool for targeting pathological and viral RNA molecules in vitro and in vivo, by suppressing gene expression through the technology of EGS (external guide sequence) and M1GS ribozymes. RNase P, according to numerous studies, has been the target of several pharmaceutical agents, including most of the mainstream antibiotics. It has been shown recently, through analytical kinetic studies that the macrolide spiramycin significantly enhances the activity of bacterial RNase P and M1 in a dose dependent manner, acting as a non-specific mixed-type activator. Until now, no other compound has been reported to induce a positive effect on RNase P activity. In the present study, the enhancement of bacterial RNase P activity by spiramycin was tested initially, and it was revealed that spiramycin does not interact with the M1 RNA molecule through ionic bonds. On the contrary, it induces a conformational change of the P10/11 structural element of M1 RNA, which is mainly responsible for substrate recognition. The above results are in agreement with the KD values determined for the ribozyme-substrate complex, in the absence or in the presence of spiramycin. Since spiramycin does not affect eucaryotic protein synthesis or eucaryotic RNase P activity, an M1GS ribozyme was constructed, in order to examine the effect of spiramycin on the ribozyme activity in vivo, using human HEK293 cells. The target of this M1GS was the transcription factor Ets2, a factor with great clinical importance, since it has been associated with several types of cancer and disease, as well as essential processes during differentiation. The important role of Ets2 in combination with the lack of data on Ets2 expression, had hitherto prevented its effective targeting by using the existing methodologies based on RNA, such as RNAi. After analysis of the secondary structure of Ets2 mRNA, two guide sequences were designed. These sequences were originally tested in trans as external guide sequences (EGS), in combination with the bacterial RNase P. The EGS303 (the number indicates the nucleotide residue cleaved by RNase P), showed an ability to induce RNase P activity in vitro. The guide sequence was then cloned and fused into the 3' end of M1 RNA ribozyme, thus producing the M1GS303 ribozyme, which was found to be effective against the target molecule. The activity of this specific ribozyme is impressively enhanced by 160% in the presence of spiramycin. In order to examine the activity of this ribozyme in vivo, the expression of the target molecule and the ribozyme were induced in E. coli cells. After 12 hours of expression, a reduced level of the target molecule was detected, because of the M1GS303 activity (about 95%). Reduction to similar levels was observed after only 4 hours from the induction of both molecules expression, in the presence of spiramycin. This observation strongly supports spiramycin’s striking positive effect on the ribozyme activity. The same set of experiments was repeated in human HEK293 cells. The activity of the ribozyme was determined qualitatively and quantitatively, by the determination of the expression of the chimeric fluorescent protein Ets2-EGFP (target molecule) at different times of expression. The M1GS ribozyme cleaves efficiently the target molecule in human cells as well in vivo, resulting in a reduction in the expression of Ets2, which is further increased in the presence of spiramycin. This result indicates the significant activation of M1GS activity in human cells, making spiramycin an important activator in using M1GS ribozymes as tools in gene silencing. The combination of improved M1GS ribozymes in the presence of spiramycin, further increases the practical utilization of this technology both in vitro and in vivo, thus achieving an even more effective suppression in gene expression.

Abstract In mice, gene targeting has become a useful tool for resolving the functions of proteins and for creating new animal models. Cre/loxP technology has been used broadly for generation of genetically modified mice. The Cre recombinase recognizes a specific DNA sequence, called loxP, and removes any DNA fragment between two loxP-sites. The activity of the Cre recombinase can be controlled spatially and temporally with cell- or tissue-specific promoters and synthetic inducing agents, such as tamoxifen or tetracycline. In this thesis, we employed tamoxifen-induced Cre recombination in vitro. Cre-ERTM mice were crossed to ROSA26LacZ reporters and Cre-recombination induced by 4OH-TM was monitored by LacZ staining. 0.5 μM 4OH-TM treatment was competent for tamoxifen-induced Cre-mediated activation of LacZ both in kidney cultures and in experimentally induced kidney mesenchymes. Wnt4 is a secreted signaling molecule, which is necessary for the development of several organs including kidney, ovary, adrenal gland, mammary and pituitary glands. Wnt4 is crucial for kidney development and conventional Wnt4-/- mice die soon after birth, likely due to renal failure. In this thesis, two different Wnt4 alleles, Wnt4EGFPCre and floxed Wnt4, were generated and analyzed to learn more about the Wnt4 functions and to apply these mouse lines to renal functional genomics. In the Wnt4EGFPCre, the EGFPCre fusion cDNA was targeted into exon I of the Wnt4 locus. EGFP-derived fluorescence was observed in the pretubular aggregates from E12.5 embryonic kidney onwards. Further characterization by crossing with the floxed ROSA26LacZ and yellow fluorescent protein (YFP) reporter lines demonstrated that in addition to the kidney, reporter expression was observed in the gonad, spinal cord, lung and the adrenal gland. The expression pattern of the Cre activity recapitulates well the known pattern of the Wnt4 gene. Time-lapse analysis in organ culture settings showed that the Wnt4 expressing cells contributed to the nephrons, some cells near the stalk of the developing ureter, as well as a number of positive supposed medullary stromal cells. In the conditional Wnt4 knock-out, loxP sites were placed to flank exons 3 to 5. The Wnt4 gene was specifically inactivated with CAGCre and Wnt4EGFPCre lines. In both of these crosses deletion of Wnt4 gene function led to impaired kidney development. In conclusion, we identified the culture conditions that can be used for the tamoxifen-induced conditional mutagenesis in tissue cultures. In addition, the created Wnt4 mouse lines serve as new useful tools for addressing the roles of Wnt4 function in diverse tissues and in different stages of development. / Tiivistelmä Hiirillä geenikohdennuksesta on muodostunut hyödyllinen väline proteiinien tehtävien selvittämisessä ja uusien eläinmallien luomisessa. Cre/loxP -tekniikkaa on käytetty laajasti muuntogeenisten hiirien tuottamisessa. Cre-rekombinaasi tunnistaa spesifisen DNA-jakson, niin kutsutun loxP:n, ja poistaa kaikki DNA-jaksot kahden loxP-sekvenssin väliltä. Cre-rekombinaasin aktiivisuutta voidaan säädellä paikallisesti ja ajallisesti solu- tai kudosspesifisillä promoottoreilla ja synteettisillä indusoivilla kemikaaleilla, kuten tamoksifeenillä tai tetrasykliinillä. Tässä väitöskirjassa hyödynsimme tamoksifeenin aiheuttamaa Cre-rekombinaatiota in vitro -kudosviljelmissä. Cre-ERTM-hiirilinja risteytettiin ROSA26LacZ-reportterilinjan kanssa, ja 4-hydroksitamoksifeenin indusoima Cre-rekombinaasin aktiivisuutta monitoroitiin LacZ–värjäyksellä. 0.5&#160;µM:n 4OH-TM konsentraatiolla LacZ-reportterigeeni saatiin aktivoitua tehokkaasti Cre-rekombinaasin avulla sekä munuaisviljelmissä että munuaismesenkyymiviljelmissä. Wnt4 on erittyvä signalointimolekyyli, jolla on keskeinen rooli useiden elinten, kuten munuaisen, munasarjan, lisämunuaisen, rintarauhasen ja aivolisäkkeen kehittymisessä. Wnt4-geenillä on ratkaisevan tärkeä rooli munuaisen kehityksessä, ja poistogeeninen Wnt4-/-hiiri kuolee pian syntymän jälkeen, todennäköisesti munuaisen vajaatoimintaan. Tässä väitöskirjatyössä tuotettiin kaksi eri Wnt4 alleelia, Wnt4EGFPCre ja konditionaalinen Wnt4. Nämä hiirilinjat analysoitiin, jotta saisimme lisää tietoa Wnt4-geenin toiminnasta ja pystyisimme soveltamaan kyseisiä hiirikantoja munuaisten toiminnan selvittämisessä. Wnt4EGFPCre-alleelissa EGFPCre-fuusio -cDNA kohdennettiin osaksi endogeenisen Wnt4-geenin ykköseksonia. Vihreän fluoresoivan proteiinin (EGFP) aktiivisuus havaittiin varhaisen munuaisen kehityksen aikana. Wnt4EGFPCre-alleelin lisäkarakterisointi reportterilinjoilla (Rosa26LacZ ja Rosa26YFP) osoitti, että Wnt4-geenin ilmentyminen havaittiin munuaisen lisäksi sukurauhasissa, selkäytimessä, keuhkoissa sekä lisämunuaisessa. Wnt4EGFPCre-alleeli ilmentyi niissä kudoksissa, joissa endogeenisen Wnt4-geenin tiedetään olevan aktiivinen. Time-lapse -analyysin avulla osoitettiin, että Wnt4-geeniä ilmentävät solut muodostavat tiettyjä rakenteita munuaisen kehityksen aikana. Wnt4-geeni ilmentyi nefroneissa, kehittyvän virtsajohtimen soluissa sekä useissa medullaarisissa stroomasoluissa. Konditionaalisessa (ehdollisessa) Wnt4 knock-out-hiirilinjassa loxP-sekvenssit sijoitettiin eksonien kolme sekä viisi ympärille. Wnt4-geenin toiminta inaktivoitiin CAGCre- ja Wnt4EGFPCre-hiirilinjojen avulla. Näissä molemmissa tapauksissa Wnt4-geenin toiminnan poistaminen johti munuaisen kehityshäiriöön. Yhteenvetona voimme todeta, että olemme tunnistaneet ne kasvatusolosuhteet, joita voidaan hyödyntää, kun halutaan aktivoida reportterigeenejä tai kehityksen kannalta tärkeitä geenejä tamoksifeenin aiheuttamaa Cre/loxP -rekombinaatiota hyväksikäyttäen kudosviljelmissä. Samoja olosuhteita ja menetelmää käyttäen voidaan myös poistaa jonkun kehityksen kannalta tärkeän geenin toiminta ja tutkia sitä kudosviljelmässä. Tuotetut Wnt4-hiirikannat ovat lisäksi uusia hyödyllisiä työkaluja, kun halutaan tutkia Wnt4-geenin toimintaa erilaisissa kudoksissa ja eri kehitysvaiheiden aikana.