Les progrès en biotechno]ogie ont permis le développement d’outils pour le transfert de gène intégratif en transgénèse, bioproduction et thérapie génique. Cependant, trois challenges majeurs doivent être relevés pour garantir un système sécurisé : l’innocuité et l’efficacité du transfert, l’intégration ciblée et contrôlée dans le génome, le niveau et la durée d’expression du transgène au cours du temps. Dans ce but, mes travaux de thèse ont consisté à tester des solutions pour améliorer la biosécurité du transposon piggyBac qui nécessite un plasmide porteur du gène d’intérêt à insérer dans le génome et une source de transposase catalysant la réaction d’intégration du transgène. Une des stratégies de ma thèse repose sur l’apport de la source de transposase sous forme d’ARN messager au lieu d’ADN afin d’améliorer la stabilité de l’intégration et de réduire les effets génotoxiques en limitant la transposase dans les cellules. Pour la première fois, la biodisponibilité de l’ARNm de la transposase et les conditions optimales d’utilisation en cellules humaines ont été déterminées pour augmenter la biosêcurité du système. Le second objectif de mes travaux consiste à améliorer l’expression du transgène en ajoutant des insulateurs connus pour s’opposer à l’extinction de l’expression des gênes. En termes de biosécurité, cette stratégie permet de réduire le nombre de copies du transgène nécessaires pour obtenir une expression suffisante. Deux candidats ont été identifiés pour améliorer l’expression du transgène. La combinaison des approches ARNrn et insulateurs est prometteuse pour sécuriser le transfert de gène médié par piggyBac et pour maintenir l’expression du gène d’intérêt. / Advances in biotechnology have enabled the development of tools for gene transfer applicable to transgenesis, bioproduction and gene therapy. But, 3 major challenges must be met to ensure a secure system: the safety and effectiveness of the transfer. the targeted and controlled integration into the genome. and the level of transgene expression over time. In this aim, my thesis project was to validate solutions to improve the biosafety of the piggyBac transposon, which requires a plasmid carrying the gene of interest to be inserted in the genome, and a source of transposase which catalyzes the transgene integration. One approach of my thesis work is to deliver the source of piggyBac transposase as an mRNA molecule instead of DNA. This strategy aims to improve the stability of the integration and reduce the genotoxic effects by limiting the transposase in the cells. For the 1st time, the bioavailability of the transposase rnRNA and the optimal conditions for its use in human cells were determined to increase the biosafety of the transposon system. The 2nd objective ofmy project is to improve the expression of the transgene by adding insulators known to counteract the transgene silencing. This strategy reduces the number of integrations required ta get a sufficient expression of the transgene and thus, improve biosecurity. Two candidates have been identified to improve transgene expression. The combination of the mRNA and insulator strategies is promising to secure the piggyBac-mediated gene transfer and to maintain the expression of the gene of interest

Due to its strength, flexibility, and biocompatibility, spider silk is a highly appealing material for applications in the medical field. Unfortunately, natural spider silk is difficult to obtain in large quantities because spiders are territorial and cannibalistic, making them impractical to farm. Synthetic spider silk proteins produced by transgenic hosts such as bacteria and goats have made it possible to obtain the quantities of spider silk needed to study it more fully and to investigate its potential uses. The spider silk proteins produced in our laboratory do not have an optimal purification method to remove all of the non-biocompatible contaminants and have not previously been tested for their biocompatibility. The first focus of this dissertation was to create goat cells that can be used to create new goats. These new goats will produce proteins that can be purified more efficiently and more completely. The second focus of this dissertation was to perform biocompatibility tests on goat-derived spider silk proteins. Prior to performing any biocompatibility tests, a method was established for removing endotoxins – an impurity that causes an immune response in the body – from the proteins. This work has shed light on areas for improvement in the silk protein purification process and laid groundwork for the production of new goat-derived proteins. These steps will help make it possible for synthetic spider silk to progress further toward becoming a viable biomaterial.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / In this work we presented the idea to perform excision assays using the piggyBac transposable element as enzyme supplier and the inverted terminal sequences of the element, both necessary for mobilization of a transposable element. Drosophila S2 cells were electroporated to perform insertion of two different plasmids in the cytoplasm of cells, a plasmid carrying the terminal inverted repeats of piggyBac element flanking a GFP gene and other with the transposase coding sequence enzyme which recognizes the terminal inverted repeats, excise of the region where the element is and insert it into another locus. This is a vector-helper system, in which a fragment is excised from a plasmid with the help of the transposase located in the other. Conventional PCR was used to verify excision events showing a 200bp amplification region where the fragment was excised and a region 3kb amplification reagion at times when the fragment was full, ie, it has not mobilized. The qPCR technique was used to quantify the excision of this fragment, carrying out comparisons of the amount of plasmid DNA recovered from the S2 cells after the end of experiment with serial dilutions of the original plasmids carrying the ITRs, which was used as standard. The results showed that the technique involving electroporation and qPCR is feasible and can be used to quantify mobilization of transposable elements. Paralleling with existing tools for this type of quantification, qPCR shows up as a very sensitive technique of detection mobilization, as well as a low cost technique budget. / Neste trabalho apresentamos a ideia de realizar ensaios de excisão utilizando o elemento transponível piggyBac como fornecedor da enzima e das sequências terminais invertidas do elemento, ambos necessários para mobilização. Células S2 de Drosophila melanogaster foram eletroporadas para que houvesse inserção de dois diferentes plasmídeos no citoplasma das células, um plasmídeo portando as repetições terminais invertidas do elemento piggyBac flanqueando um gene GFP e o outro com a sequência codificadora da enzima transposase, a qual reconhece as repetições terminais invertidas e excisa o elemento da região onde está inserido, num sistema vector-helper, em que um fragmento é excisado de um plasmídeo com ajuda da transposase localizada no outro. PCR convencional foi usado para verificar os eventos de excisão, mostrando uma região de amplificação de 200pb nos casos de excisão do fragmento e uma região amplicada de 3kb, nas ocasiões em que o fragmento ficou inteiro, ou seja, não foi mobilizado. A qPCR foi utilizada para quantificar a excisão desse fragmento, realizando comparações da quantidade de DNA plasmidial recuperado das células S2 após o término do experimento com diluições em série do plasmídeo com as ITRs, que foi utilizado como standard. Os resultados mostraram que a técnica envolvendo eletroporação e qPCR é exequível e pode ser utilizada para quantificar mobilização de elementos transponíveis. Fazendo um paralelo com as ferramentas já existentes para esse tipo de quantificação, qPCR mostra-se como uma técnica bastante sensível de detecção de mobilização, bem como uma técnica de baixo custo orçamentário.

The childhood eye cancer retinoblastoma originates from the retina and its development is initiated while the foetus is in the uterus. Retinoblastoma has a reported incidence of 1 in 15-18 000 live births, and approximately 90% of all patients are diagnosed before the age of 5. The occurrence of retinoblastoma is usually detected by the parents and the most frequent symptoms are leukocoria (white pupillary reflex), strabismus (squinting) or if the child complains of visual problems. Retinoblastoma is diagnosed by examination under anaesthesia and documentation by RetCam. It is treated with various cytostatic agents, or by laser. If the treatment is unsuccessful, or there is a risk that the tumour cells will spread and form metastases, the eye is removed. Previous studies have indicated that the cell type from which the tumour arises, the cell-of-origin, may be the cone photoreceptors and/or their immediate interneuron, the horizontal cells. Determining the cell-of-origin for retinoblastoma is an important goal, however, understanding the molecular mechanisms that distinguish the photoreceptors and the horizontal cells from the other retinal cells may prove just as important for understanding this disease. The aim of my project has been to develop, optimise and validate methods to label, map and target expression to photoreceptors and horizontal cells in the chicken embryonic retina. We have successfully established several methods that test the expression pattern of conserved, regulatory DNA sequences, and have performed short- and long-term expression of various genes that have been reported to be involved in cell cycle regulation and cell fate determination. One of my most important findings was that a region from the RXRγ gene allowed us to specifically target the photoreceptors and horizontal cells. Our previous knowledge, together with the newly established tools, puts us an important step closer towards understanding the development and behaviour of the retinal photoreceptors and horizontal cells, however, further studies are of course needed.

Primordial germ cells (PGCs) are the precursors of the germ cell lineage that eventually differentiate into mature spermatozoa and oocytes. Although present throughout the animal kingdom, the specification and migration of PGCs differs widely between species. In vertebrates, avians are evolutionary divergent from mammals and therefore allow a comparative system in which to study germ cell development in higher organisms. Unlike mouse, PGCs can be isolated from the chicken embryo, expanded and cultured long term in vitro. Analysis of these cells showed that cultured chicken PGCs maintain the characteristics of their in vivo counterparts, including the expression of key germ cell specific markers and cell surface adhesion proteins, and thus, are an ideal system to study germ cell biology. Further characterisation revealed that an avian homologue of the zinc finger transcription factor PRDM14, essential for the specification of the mammalian germ cell lineage, was expressed in chicken PGCs. cPRDM14 was found to be expressed in PGCs in vitro and in vivo from early developmental stages until expression is lost by embryonic day 10 and subsequently re-expressed in the adult testis. The expression of cPRDM14 suggested that this gene may play a conserved role in the avian germ cell lineage. To investigate the function of cPRDM14, a novel single piggyBac transposon vector containing a reverse tetracycline activator protein and a tetracycline response element-regulated promoter was developed. Testing of the integrated transposon revealed that expression was tightly regulated and it was possible to conditionally express one gene product whilst simultaneously reducing the expression of a second gene, both in vitro and in vivo. This vector system was fully functional in PGCs, and was used to create transgenic founder chickens capable of having gene expression manipulated in germ cells at various developmental stages. Transgenic offspring were produced and the transgene was inducible at early developmental stages in the G1 animals. The un-induced transgene proved to be toxic to early embryos so a transgenic line of birds could not be produced. The inducible transposon was used to knockdown cPRDM14 expression in chicken PGCs. Knockdown of this gene led to reduced PGC numbers and increased cell death, both in vitro and in ovo. Expression of the pluripotency factor cNANOG was also significantly reduced which may explain the increased cell death. The knockdown of cPRDM14 also led to an increased susceptibility of PGCs to spontaneously de-differentiate to pluripotent embryonic germ cells (EGCs). cPRDM14 knockdown PGCs exhibited elevated levels of phosphorylated ERK, a target of the FGF signalling pathway. It was possible to prevent de-differentiation of the knockdown PGCs by removing ectopic FGF from the media. Furthermore, a sustained high level of FGF signalling in the media was sufficient to drive the de-differentiation of control PGCs to EGCs, suggesting that increased FGF signalling was key to the de-differentiation process. Extensive epigenetic remodelling of mouse PGCs occurs during embryonic development and PRDM14 was shown to be involved in this process. Chicken PGCs in vitro, contain several key histone modifications (H3K4me3, H3K9me2 and H3K27me3) and are 5-methyl cytosine (5-mC) positive. Immunohistochemical analysis of these markers in PGCs, at various stages during early embryonic development, suggests that these cells do not undergo the extensive epigenetic remodelling found in their mammalian counterparts. In contrast to the mouse germ cell lineage, knockdown of cPRDM14 in cultured PGCs had no noticeable effect on the epigenetic status of chicken PGCs. Together these results demonstrate that cPRDM14 is essential for the survival and maintenance of germ cell identity in chicken PGCs, but may not be critical for maintaining the epigenetic status of these cells.

Transgenic manipulation of exogenous and endogenous gene expression in human embryonic stem cells (hESCs) is a powerful approach to decipher the genetic pathways dictating their developmental fate. Presently used genetic tools face limitations including leakiness in inducibility of expression, epigenetic silencing in long-term cell culture, low genomic integration efficiencies, small genetic cargo limit and lack of high-throughput cloning capabilities. To overcome these limitations, I have constructed R4-Integrase and piggyBac transposon genetic vector systems for stable transgene overexpression and knockdown in hESCs. Preliminary functional testing of the piggyBac vector system in HEK 293T and hESCs demonstrated vector inducibility as well as successful overexpression and knockdown of pluripotency factor OCT4. Concurrently, a cost-effective and high efficiency method for chemical transfection of hESCs was developed. Exogenous overexpression and knockdown of transcription factors in hESCs will aid in the elucidation of gene regulatory networks controlling pluripotency and developmental fate.

Transgenic manipulation of exogenous and endogenous gene expression in human embryonic stem cells (hESCs) is a powerful approach to decipher the genetic pathways dictating their developmental fate. Presently used genetic tools face limitations including leakiness in inducibility of expression, epigenetic silencing in long-term cell culture, low genomic integration efficiencies, small genetic cargo limit and lack of high-throughput cloning capabilities. To overcome these limitations, I have constructed R4-Integrase and piggyBac transposon genetic vector systems for stable transgene overexpression and knockdown in hESCs. Preliminary functional testing of the piggyBac vector system in HEK 293T and hESCs demonstrated vector inducibility as well as successful overexpression and knockdown of pluripotency factor OCT4. Concurrently, a cost-effective and high efficiency method for chemical transfection of hESCs was developed. Exogenous overexpression and knockdown of transcription factors in hESCs will aid in the elucidation of gene regulatory networks controlling pluripotency and developmental fate.

Infectious diseases that spread from person-to-person and continent-to-continent are a cause for concern for any health entity. One such disease is malaria, a mosquito-borne infection instigated by the protozoan parasite, Plasmodium falciparum. Hundreds of millions of people are affected annually and it is responsible for nearly 1 million deaths. It is the most fatal species causing malaria and proliferates in human red blood cells with a life cycle occurring every 48 hours. At this time, the parasite’s late stage form or schizont bursts from the erythrocyte releasing immune-inducing particles and infective forms (merozoites) into the bloodstream. The merozoites go on to infect other red blood cells as human immunity leads to fever. Fever is a hallmark symptom of malaria and effectively inhibits the growth of late stage parasites. Plasmodium still manages to complete its life cycle as early stages or rings are not affected by febrile temperatures. It is this facet of parasite biology that prompts our research into identifying genetic factors associated with fever. The parasite’s response under elevated body temperature may offer further insight into its adaptive mechanism. A heat shock assay was developed in order to simulate fever in vitro. Mutant parasite cultures were subjected to 41°C for 8 hours and returned to normal body temperature or 37°C for the remainder of the life cycle. The piggyBac mutagenesis system allows for the evaluation of phenotypes associated with a particular genotype as the transposon inserts randomly into the gene. This often leads to changes in function that may cause delays in invasion or attenuation of growth. Determining the genes responsible for these phenotypes would be a great advantage to the field of drug discovery. Collaborative efforts to develop vaccines and new antimalarial drugs are underway as resistance to current methods of treatment is on the rise. Such circumstances require new technologies for detecting novel drug targets or pathways in the parasite that can be significantly affected by these therapeutics. QISeq is a next generation sequencing tool that identifies genes with a particular phenotype that may alter intraerythrocytic development of P. falciparum. This technique was utilized in our study to confirm the heat shock phenotype with a high-throughput approach. The genomic DNA of pooled parasite cultures was sequenced to reveal those mutants sensitive and/or resistant to febrile temperature exposure. Through bioinformatics analyses, functional associations between genes can be made that lead to biological pathways of interest for therapeutic research.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19899号 / 農博第2182号 / 新制||農||1043(附属図書館) / 学位論文||H28||N5003(農学部図書室) / 32976 / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 今井 裕, 教授 久米 新一, 教授 廣岡 博之 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Whole genome sequencing (WGS) projects for model mammalian organisms exposed the magnitude to which transposable elements (TEs) have contributed to DNA content, but led to inferences on repeat composition and activity patterns which do not capture the true diversity within Mammalia. Understanding of the evolutionary importance of TEs and the development of TEs for biological applications are hindered when considering only the data gathered from a limited sampling of model organisms. The research presented here begins with analysis of a mouse lemur WGS dataset, revealing an exception in the primate lineage to the generalizations garnered from model organisms. A recently active TE was uncovered with evidence of horizontal transfer involving a primate and another mammal; this discovery may lead to a useful transfection vector with known efficacy in mammalian cells. Furthermore, an opportunity to observe atypical patterns of TE diversification within a mammalian family was achieved through survey sequencing and comparative analyses of five vespertilionid taxa and a phyllostomid outgroup. A potential inverse relationship between the two classes of TEs was tentatively described, and further explored by an in-depth analysis of the dominant TE family from each class in a species with WGS data available. The interplay of the two classes could not previously be investigated in mammals, due to ~40 million years of Class II TE inactivity in model organisms. Through exploration of activity patterns of both classes, this study provides insight on the relationship between TEs and their host.