Although the understanding of genetic predisposition to prostate cancer (PCa) has been improved through genome-wide association studies (GWAS), little is known about the biological implication of germline variants residing in coding or non-coding regions in cancer development and progression. Our hypothesis is that inherited variants may predispose to specific early recurrent genomic events observed in PCa adenocarcinomas, possibly in the context of variable androgen receptor (AR) signaling that changes during a man’s lifetime. Recent in silico analysis by our group on potential association between germline variants and PCa specific somatic lesions identified a non-coding polymorphic regulatory element at the 7p14.3 locus associated with DNA repair and hormone regulated transcript levels and with an early recurrent prostate cancer specific somatic mutation in the Speckle-Type POZ protein (SPOP) gene (OR=5.54, P=1.22e-08) in human prostate tissue data. In order to functionally characterize the polymorphic 7p14.3 locus (rs1376350, single nucleotide polymorphism, G>A), we set up to establish isogenic cell lines harboring the minor allele by using the CRISPR/Cas9 system. In parallel, CRISPR/Cas9 system was used to knock out different portion of the region encompassing the 7p14.3 variant and to eliminate transcription factors (TFs) binding sites that were identified from previous in silico analysis (i.e. AR and CCAAT/Enhancer Binding Protein (C/EBP) beta (CEBPβ)). The transcriptomes of edited pools and edited single clones from macrodeletion (731 bp), microdeletion (50 bp) and alterations of TFs binding sites were analyzed and compared to the transcriptomes of isogenic cells heterozygous (A/G) and homozygous (A/A) for the minor allele A of the risk variant rs1376350 (with or without AR overexpression). These data identified a set of genes scattered throughout the genome with the same pattern of deregulation suggesting the implication of the variant on the regulation of genes residing in different chromosomes. Additionally, ChIP-qPCR experiments for histone modification supported the identification of the 7p14.3 locus with enhancer activity. Furthermore, ChIP-qPCR of histone mark associated with transcriptional activation or repression in isogenic cells harboring the minor allele A upon AR overexpression showed that the activity of the locus is higher for the minor allele A compared to G, independently from AR activation. Despite the limitations of our model and the current lack of validation in other cells, we confirmed that some of the differentially expressed genes that emerged from the comparative analysis of edited cells are deregulated in human normal and tumor prostate samples as well. This work is a proof of concept of germline predisposition to molecularly distinct cancer subclasses and has the potential to nominate new mechanisms of cancer development. Future work aims to elucidate the mechanisms implicated in the deregulation of the transcriptome by combining the information obtained until now with potential new players that we expect to identify by Mass Spectrometry experiments. To clarify the link between the 7p14.3 variant and the somatic mutations in SPOP, we plan to express mutant SPOP in isogenic cells harboring the minor allele and to asses DNA damage response upon overexpression or silencing of TFs binding at and around the rs1376350 variant. My work is an example of how the CRISPR/Cas9 system can be used to develop a technical framework with convergent approaches to functionally characterize polymorphic regulatory regions including but not limited to the establishment of isogenic cells upon single nucleotide editing.

von Willebrand factor (VWF) protein acts in the intrinsic coagulation pathway by stabilizing FVIII from proteolytic clearance and at the site of injury, by promoting the adhesion and aggregation of platelets to the exposed subendothelial wall. von Willebrand disease (VWD) results from quantitative and qualitative deficiencies in VWF protein. The variability expressivity in phenotype presentations is in partly caused by the action of modifier genes. Zebrafish has been used as hemostasis animal model. However, it has not been used to evaluate VWD. Here, we report the development of a heterozygote VWF mutant zebrafish using the genome editing CRISPR/Cas9 system to screen for modifier genes involved in VWD. We designed CRISPR oligonucleotides and inserted them into pT7-gRNa plasmid. We then prepared VWF gRNA along with the endonuclease Cas9 RNA from Cas9 plasmid. We injected these two RNAs into 1-4 cell-stage zebrafish embryos and induced a mutation in VWF exon 29 of the zebrafish with a mutagenesis rate of 16.6% (3/18 adult fish). Also, we observed a germline transmission with an efficiency rate of 5.5% (1/18 adult fish). We obtained a deletion in exon 29 which should result in truncated VWF protein.

The neurotransmitter dopamine (DA) plays a critical role in regulating cognition, behavior and physiology in humans. Imbalances in DA or damage to the dopaminergic (DAnergic) system can be consequential to neurological health and lead to the progression of psychiatric and neurodegenerative disorders that include but are not limited to schizophrenia and Parkinson’s disease (PD). PD, in particular, is associated with debilitating motor symptoms that result following a considerable loss of midbrain DAnergic neurons. This loss is likely correlated to a combinatory insult of environmental exposures and genetic predisposition, as the majority of cases are idiopathic in nature. To date there remains to be a curative treatment, thus much research has been done to generate models of sporadic PD through the use of neurotoxic exposures in addition to the search for plant-derived chemicals that confer neuroprotection prior to the onset of symptoms to improve the quality of life for those at risk. Here, we established a model to mimic pathologies observed in sporadic PD using both larval and adult zebrafish. The larval model examined the neurotoxic impact of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 1-methyl-4-phenyl-pyridinium (MPP+), 6-hydroxydopamine (6-OHDA), rotenone, and paraquat to delineate the optimal compound that exerts the largest degree of diencephalic DAnergic cell death and motor perturbance using Tg(dat:eGFP) transgenic zebrafish. Using this model, we also showed that between ascorbic acid (AA), ferulic acid (FA) and vanillic acid (VA), pretreatment of FA elicits that largest neuroprotective effect against MPTP-induced oxidative stress and neurodegeneration. The optimization of a reliable adult model to investigate mitochondrial impacts in vivo was then addressed through introducing MPTP into the cerebroventricular fluid of Tg(dat:tom20 MLS:mCherry) transgenic zebrafish. Gene expression and immunostaining data suggest that MPTP induces DAnergic mitochondrial fragmentation through mitophagy activation. Moreover, we sought to examine the genetic influence over DAnergic production and disorders by targeting nr4a2 paralogs for CRISPR-Cas9 mediated mutagenesis. Despite a similar deleterious effect observed in DAnergic populations, nr4a2a and nr4a2b mutants each possess variable effects on neurotrophins, metabolism, other neurotransmitters and behavior. nr4a2a mutants more closely resemble PD pathologies, whereas nr4a2b mutants exhibit phenotypes reminiscent of psychiatric disorders. Throughout DAnergic regeneration, nr4a2a was also shown to mimic shha expression patterns suggestive of a predominant role over nr4a2b in differentiation. Further gene expression data may also indicate that notch1a drives the proliferative stages of DAnergic progenitors prior to the shift to shha signaling for differentiation. In sum, we believe the sporadic and genetic models of DA deficiencies offer an opportunistic tool to study molecular mechanisms of DAnergic regeneration, potential therapeutics and to gain a better understanding of mitochondrial influence in neurological pathologies.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第23246号 / 農博第2453号 / 新制||農||1084(附属図書館) / 学位論文||R3||N5336(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 植田 充美, 教授 白井 理, 教授 栗原 達夫 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

No description available.

Myelodysplastic syndromes (MDS) encompass a heterogeneous group of age-related hematopoietic disorders characterized by ineffective and incomplete hematopoiesis leading to an increased risk of acute myeloid leukemia (AML). The development of accurate and easily used in vitro models is crucial for understanding the pathogenesis of MDS and identifying potential therapeutic targets. Induced pluripotent stem cells (iPSCs) can be used to study MDS due to their ability to differentiate into any cell type depending on the environment. The main limitation is that the blood progenitors produced by iPSCs are of a fetal state, which hinders modeling of MDS, a disease of older adulthood. This study aimed to optimize the maturation state of blood progenitors derived from iPSCs by induction of the micro-RNA let-7, which, we hypothesize will increase the maturation and adult phenotypic state of hematopoietic progenitors. iPSC lines were generated from healthy controls and samples containing the SRSF2 mutation, a common mutation in MDS, containing a doxycycline-inducible, stabilized let-7 transgene. A stepwise differentiation efficiently drove the iPSCs toward hematopoietic progenitors and, subsequently, other mature lineages. The hematopoietic progenitors were characterized by assessing the expression of specific cell surface markers and functional properties using flow cytometry, colony-forming assays, and multi-lineage differentiation abilities. These findings demonstrate the potential of using iPSC engineering to create a novel model for MDS and other age-biased disorders by inducing let-7 expression in iPSCs and, when differentiating them, exposing them to doxycycline to promote an adult cell phenotype. This approach offers a valuable potential tool for elucidating the molecular mechanisms underlying these disorders and exploring potential therapeutic interventions. / 2026-03-13T00:00:00Z

The chicken is a classic model organism that has provided key insights into embryonic development. Chicken embryos can be directly manipulated and observed during development while retaining the potential to reach adulthood. Despite this benefit, the utility of the chicken in studying development has been limited by the difficulty of introducing genetic changes to the genome. The recent development of cell culture conditions for chicken primordial germ cells (cPGC) has made it feasible to produce transgenic chickens, but there is still a lack of tools for introducing genetic modifications into cPGCs. Recombinase Mediated Cassette Exchange (RMCE) is a technique that has been utilized in traditional genetic systems to generate multiple alleles at a given locus but has not yet been adapted to the chicken. In order to use RMCE in the chicken, we inserted Lox sites into cPGC using CRISPR/Cas9. We targeted the ovalbumin locus and potential genomic safe harbor sites (GSH) identified using genomic data. We performed RMCE to exchange green fluorescent protein (GFP) into these loci. We observed RMCE efficiency as less than 1% at each loci. We then designed a system using a drug inducible Caspase 9 (iCasp9) to select for cells that underwent cassette exchange. This method enabled us to obtain a population of 100% edited cells. We anticipate that this tool will increase the utility of the chicken as a model organism, livestock, and bioreactor.

The CRISPR/Cas9 system has become the predominant tool for genome editing. Targeted modifications can be introduced while repairing double strand breaks (DSBs), induced by the CRISPR/Cas9 system. The DSB is repaired by either non-homologous end joining (NHEJ) or homologous recombination (HR), and the repair is commonly processed through NHEJ because it is the dominant repair pathway in most cell types. The goal of this study is to modulate DNA repair system of somatic cells to increase the frequency of homology-directed repair (HDR) through HR by chemical treatment and the frequency of NHEJ by serum starvation. CRISPR/Cas9 systems targeting RAG2 gene and donor DNA to replace endogenous RAG2 were transfected into porcine fetal fibroblast (PFF) cells and the cells were treated with various chemicals that were known to inhibit NHEJ or stimulate HR. Among the chemical treated groups, cells treated with thymidine showed an average of 5.85-fold increase in HDR compared to the control group; the difference ranged from 1.37 to 9.59. There was no positive effect on the frequency of HDR after treating transfected cells with other chemicals. Placing PFFs under low amount of serum (serum deprivation) could enrich the cells in G0/G1 phase, but there was little difference in the frequency of NHEJ. Our results indicate that modulating DNA repair pathways during CRISPR/Cas9-mediated gene targeting could change the outcome of the targeted events. / Master of Science / The CRISPR/Cas9 system is the newest generation of genetic engineering tool for genome editing. Genetic modifications can be introduced while repairing double strand breaks (DSBs) on DNA, induced by the CRISPR/Cas9 system. The DSB is repaired by either non-homologous end joining (NHEJ) or homologous recombination (HR), and the repair is commonly processed through NHEJ because it is the dominant repair pathway in most cell types. The goal of this study is to modulate DNA repair system of somatic cells to increase the frequency of homology-directed repair (HDR) through HR by chemical treatment and the frequency of NHEJ by serum starvation. Among the chemical treated groups, cells treated with thymidine showed an average of 5.85-fold increase in HDR compared to the control group; the difference ranged from 1.37 to 9.59. There was no positive effect on the frequency of HDR after treating transfected cells with other chemicals. Placing pig fibroblast under low amount of nutrient could enrich the cells in G0/G1 phase of cell cycle, but there was little difference in the frequency of NHEJ. Our results indicate that modulating DNA repair pathways during CRISPR/Cas9-mediated gene targeting could change the outcome of the targeted events.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第25316号 / 農博第2582号 / 新制||農||1104(附属図書館) / 京都大学大学院農学研究科農学専攻 / (主査)教授 田尾 龍太郎, 教授 田中 義行, 准教授 中野 龍平 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Les furocoumarines sont des composés phénoliques impliqués dans la défense contre les herbivores. Ces molécules sont majoritairement décrites dans quatre familles botaniques, notamment les Rutaceae, dont font partie les agrumes. Ces molécules sont phototoxiques ce qui peut poser des problèmes pour leur utilisation comme par exemple en cosmétique ou en phytothérapie. D’autre part, en cas d’ingestion par exemple via la consommation de jus de certains agrumes, elles ont responsables de l’inhibition d’enzymes de détoxication comme le CYP3A4 humain. Cela peut conduire à des surdosages médicamenteux connus sous le nom d’Effet Pomelo. Ce travail de thèse a consisté à réfléchir et à développer, des outils qui permettront de générer de manière ciblée des variétés de pomelo qui ne produisent plus de furocoumarines. Nous avons abordé l’ensemble des étapes essentielles pour la mise en place d’une stratégie global : i) des méthodes reproductibles ont été développées pour la production de protoplastes et de cultures cellulaires de pomelo Star Ruby ; ii) des conditions de transformation de protoplastes par électroporation ont également été mises au point ; iii) finalement, pour inhiber de manière spécifique la voie de biosynthèse des furocoumarines, nous avons choisi de mettre en œuvre une approche d’édition de génome en utilisant une méthodologie CRISPR/Cas9. La mise au point de la méthode a été réalisée avec un gène codant pour une umbelliferone 6-dimethylallyl transférase. Les résultats obtenus indiquent que la stratégie est envisageable. Pour renforcer la stratégie CRISPR/Cas9, nous avons mis en œuvre une démarche d’identification de gènes cibles additionnels. En utilisant une approche de data mining de bases de données génomiques et transcriptomiques nous avons identifié 18 séquences candidates, potentiellement impliquées dans la voie de biosynthèse des furocoumarines. L’expression hétérologue des protéines correspondantes et leur caractérisation fonctionnelle a permis de montrer que CYP706J12 est en mesure de métaboliser l’hérniarine, une coumarine. Ce résultat apporte des éléments pour émettre des hypothèses sur l’évolution convergente de la synthèse des coumarines et des furocoumarines chez les végétaux supérieurs. / Furanocoumarins are phenolic compounds involved in defense against herbivores. These molecules are mainly described in four botanical families. Rutaceae, one of those families, includes Citrus species. Furanocoumarins are phototoxic compounds, which can be problematic for their use in cosmetics or in phytotherapy. Furanocoumarin ingestion via citrus juice consumption, may inhibit human enzymes of detoxification, such as human CYP3A4. This can lead to drug overdoses known as the “Grapefruit Juice Effect”. This work consisted in the development of tools that will allow to generate new varieties of pomelo that no longer produce furanocoumarins by targeted genome edition. We have covered the essential steps for the implementation of a global strategy: i) reproducible methods have been developed for the production of protoplasts and cell cultures of Star Ruby grapefruit; ii) conditions for protoplast transformation by electroporation have also been developed; iii) finally, to specifically inhibit the furanocoumarin biosynthetic pathway, we chose to implement a genome editing approach using a CRISPR / Cas9 methodology. The development of the method was carried out with a gene encoding umbelliferon 6-dimethylallyltransferase. The results obtained indicate that the strategy is feasible. To strengthen the CRISPR / Cas9 strategy, we implemented a method to identify additional target genes. Using a data mining approach of available genomic and transcriptomic databases we identified 18 candidate sequences potentially involved in the furanocoumarin biosynthetic pathway. Heterologous expression of the corresponding proteins and their functional characterization made it possible to show that CYP706J12 is able to metabolize herniarin (a coumarin). This result provides elements to hypothesize about the convergent evolution of coumarin and furanocoumarin synthesis in higher plants.