Thesis (doktorsexamen)--Göteborgs universitet, 1986. / Extra t.p. with thesis statement inserted. Includes bibliographical references (p. 272-275).

Habilitationsschrift--Freie Universität, Berlin, 1967. / Bibliography: p. [258]-288.

Thesis--Zürich. / Vita. Includes index.

Thesis--Heidelberg. / Vita. Bibliography: p. [75]-79.

Large animal species, such as cattle, sheep and pigs, have great potential value to scientific research. This is due to their physiological similarity to humans, meaning they make excellent disease models in addition to their inherent agricultural value. However, the efficiency with which such animals can be created has been a critical barrier to their use in bioscience. Research into creating genetically modified large animals has not progressed as rapidly as research on smaller mammals, such as mice, for two main reasons. Firstly, technologies such as pluripotent stem cells, which are well established in rodents, are lacking for large animals. Secondly, large animals cannot produce as many offspring within a given time frame as mice or rats. This, combined with the low efficiencies and lack of precision of current transgenic methods, severely reduces the likelihood of obtaining an animal with a desired genotype within a viable amount of time. Recently, new tools known as ’genome editors’ have been developed to facilitate genetic modification of animals. The vastly enhanced efficiency of these editors in comparison to previous gene targeting methods, combined with the fact that genome editors do not require marker genes to be used, mean that creating genetically modified livestock is now far more feasible. This thesis investigates whether two types of genome editor, TALENs and CRISPR/Cas9, can be used to produce genetically modified large animals for a range of applications. Genome editors were combined with interspecific blastocyst complementation techniques to produce chimeric rodents where the haematopoietic system is partially or fully derived from the donor cells. This work was carried out with a long-term aim of producing chimeric animals which could produce human organs suitable for transplantation. Initial blastocyst complementation experiments were carried out by injecting murine ESCs into wildtype rat blastocysts. One animal resulting from these injections showed chimerism in several tissues. Further experiments were carried out using rat ESCs and mouse blastocysts which were either Runx1-/- or Rag1-/-, however no additional chimeras were identified. In addition to these experiments, TALENs and sgRNAs were designed against Runx1 and Rag1 in sheep and pigs in order to create a large animal model for future blastocyst complementation experiments. Increasing animal productivity is a key step in meeting the demands of an increasing global population and tackling future food insecurities. TALENs and sgRNAs for use in the CRISPR/ Cas9 system were created to target the myostatin gene in sheep. Myostatin is a negative regulator of muscle growth and animals which acquire natural inactivating mutations in both myostatin alleles exhibit a well-characterised double-muscled phenotype, where total muscle mass is about 20% greater than that of a wildtype animal. Embryo microinjections were carried out using both types of genome editor and two edited lambs were produced, one from each editor. The TALEN-edited lamb was mosaic for a deletion of arginine 283 which, upon further analysis of the muscle, did not appear to cause a significant phenotype. The CRISPR-edited lamb was heterozygous for a 20bp deletion, causing the formation of a premature stop codon and severe truncation of the mature myostatin protein. Based on data from other myostatin-knockout animals, including the Belgian Blue cattle breed, this truncated protein is not thought to be functional. To determine if this is indeed the case, the CRISPR-edited lamb is now part of a breeding programme to amplify the edited allele. To discover if genome editors could be applied to create disease-resistant animals, the project focused on foot and mouth disease. Through a literature search and bioinformatic analysis of the bovine and porcine proteomes, three host genes which are cleaved by the virus were identified; eIF4A1, eIF4G1 and IKBKG. TALENs were designed to bind and cut at the FMDV protease cleavage sites in all three genes in order to disrupt protease cleavage and reduce viral replication by slowing viral disruption of the host translation and innate immune response pathways. Although none of the TALENs showed any signs of activity, this thesis sets out some potential directions for future work. In conclusion, this thesis shows that, despite some technical issues, genome editors are a promising technology for the creation of genetically modified livestock.

As the cultural upheaval of the `60s fought its way into the `70s, Bill Talen began his career first as a poet, hitchhiking the interstate highways from the Midwest to the Coasts eagerly engaging the literary, intellectual, and artistic opportunities offered by those cultural venues. He settled in San Francisco where he earned an M.A. in Theatre Arts and joined with friends to open a theatre, "Life on the Water." Here Talen met Sydney Lanier, a minister, who became his lifelong mentor and champion. Lanier recognized in Talen a bold presence which accompanies successful preachers and elevates their sermons. He promoted and supported Talen's move to New York City where Talen fully embraced his role as Reverend Billy and directed the full might of his talents against consumerism--especially Michael Bloomberg's socio-economic goals for the City. Eventually, Talen's critique came to challenge foreign policies that promote corporatism, environmental decline, and the global homogenization of culture. Talen's body of work is extensive and two strong threads run through it that are exemplary. One evidences a complete and purposeful disregard for any artistic borders, especially the edgy land between acting and not-acting, including the tiny gradients as one merges into the other. Talen's recognition of the porosity of borders likely facilitated his willing assimilation of his character, Reverend Billy, into his own daily life and persona, until the two merged, endowing Talen, the performance artist, with the skills and insights of a spiritual leader. The second thread is simply Talen's life's journey from reluctant performer of a religious role, to the willing engagement of that role, and finally the adoption of spiritual responsibility, eventually forming a church and a religion based on activism and a strong commitment to environmental causes. The performance artist became Reverend; the Reverend was born to act. This merging of talents, goals, and dreams created a character who would run for public office. It created a performance artist who would wed lovers, baptize new congregants, and console the grieving.

Aedes aegypti (Ae. aegypti) is one of the most important vectors of dengue, chikungunya and yellow fever viruses. The use of chemical control strategies such as insecticides is associated with problems including the development of insecticide resistance, side effects on animal and human health, and environmental concerns. Because current methods have not proven sufficient to control these diseases, developing novel, genetics-based, control strategies to limit the transmission of disease is urgently needed. Increased knowledge about mosquito-pathogen relationships and the molecular biology of mosquitoes now makes it possible to generate transgenic mosquito strains that are unable to transmit various parasites or viruses. Ae. aegypti genetic experiments are enabled, and limited by, the catalog of promoter elements available to drive transgene expression. To find a promoter able to drive robust expression of firefly (FF) luciferase in Ae. aegypti embryos, an experiment was designed to compare Ae. aegypti endogenous and exogenous promoters. The PUb promoter was found to be extremely robust in expression of FF luciferase in different stages of embryonic development from 2-72 hours after injection. In subsequent experiments, transformation frequency was calculated using four different promoters (IE1, UbL40, hsp82 and PUb) to express the Mos1 transposase open reading frame in Mos1-mediated transgenesis. Germline transformation efficiency and size of transgenic cluster were not significantly different when using endogenous Ae. aegypti PUb or the commonly used exogenous Drosophila hsp82 promoter to express Mos1 transposase. This study also describes the development of new tools for gene editing in the Ae. aegypti mosquito genome and the use of these tools to design an efficient gene drive system in this mosquito. Homing endonucleases (HEs) are selfish elements which catalyze double-stranded DNA (dsDNA) breaks in a sequence-specific manner. The activities of four HEs (Y2-I-AniI, I-CreI, I-PpoI, and I-SceI) were investigated for their ability to catalyze the excision of genomic segments from the Ae. aegypti genome. All four enzymes were found to be active in Ae. aegypti; however, the activity of Y2-I-AniI was higher compared to the other three enzymes. Single-strand annealing (SSA) and non-homologous end-joining (NHEJ) pathways were identified as mechanisms to repair HE-induced dsDNA breaks. TALE nucleases (TALENs) are a group of artificial enzymes capable of generating site-specific DNA lesions. To examine the ability of TALENs for gene editing in Ae. aegypti, a pair of TALENs targeted to the kmo gene were expressed from a plasmid following embryonic injection. Twenty to forty percent of fertile G0 produced white-eyed progeny which resulted from disruption of the kmo gene. Most of these individuals produced more than 20% white-eyed progeny, with some producing up to 75%. A small deletion of one to seven bp occurred at the TALEN recognition site. These results show that TALEN and HEs are highly active in the Ae. aegypti germline and can be used for gene editing and gene drive strategies in Ae. aegypti. / Ph. D.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第21692号 / 医科博第96号 / 新制||医科||7(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 渡邉 大, 教授 伊佐 正, 教授 村井 俊哉 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Le xeroderma pigmentosum (XP) est une maladie génétique rare caractérisée par une hypersensibilité aux ultraviolets (UV) et une forte incidence des tumeurs cutanées. Les cellules des patients XP sont incapables d’éliminer les lésions induites dans l’ADN par les UV en raison d’un dysfonctionnement du mécanisme de réparation par excision de nucléotides (NER). Plusieurs groupes de complémentation ont été identifiés dans le syndrome XP, parmi lesquels le groupe XP-C représente la majorité des patients à travers le monde.Au cours de mon travail de thèse, j’ai développé un modèle de correction ciblée par recombinaison homologue (RH) d’une délétion de deux nucléotides au niveau de l’exon 9 du gène XPC aboutissant à l’apparition prématurée d’un codon stop. Afin de stimuler la RH, deux types de nucléases ingéniérées sont utilisées : les méganucléases et les TALENs. J’ai observé que la méthylation de la séquence ciblée pouvait affecter l’activité de celles-ci et donc l’efficacité du ciblage de gène. Cependant, deux approches ont été développées pour résoudre ce problème : l’utilisation d’un agent déméthylant (5-aza-2’-désoxycytidine (5azadC)) ou la création d’une endonucléase insensible à la méthylation. L’utilisation des méganucléases en combinaison avec la 5azadC a permis de stimuler la fréquence de coupure de presque 20 fois dans des fibroblastes XPC et la TALEN modifiée permet une augmentation de 40 fois. Avec ces deux stratégies j’ai obtenu des événements de correction génétique par introduction d’une matrice de réparation dans le locus ciblé avec une fréquence proche de 3%. La caractérisation des clones corrigés avec la TALEN XPC montre la correction génomique des deux nucléotides dans l’exon 9, une restauration de l’expression de la protéine XPC et une résistance cellulaire après irradiation UV traduisant le rétablissement des fonctions de la NER. Cette étude représente la première preuve de correction génétique de cellules déficientes en protéine XPC en utilisant une approche ciblée. / Xeroderma pigmentosum (XP) is a rare inherited genetic disorder characterized by an UV hypersensitivity and a severe predisposition to skin cancers. Cells from XP patients are deficient in nucleotide excision repair (NER) of UV‐induced DNA lesions. Several complementation groups have been identified in the XP syndrome and the XP-C group represents the majority of XP patients around the world. During my PhD work, I developed a model of targeted correction by homologous recombination (HR) in order to correct a deletion of two nucleotides in the ninth exon in XPC gene leading to a premature stop codon. To stimulate HR, I used two types of engineered endonucleases : meganucleases and TALEN. I observed that the target methylation status could affect the endonuclease activities and therefore XPC gene correction. Nervertheless, I developed two approaches to overcome this methylation sensitivity : use of a demethylating agent (5-aza-2-deoxycytidine (5azadC)) or a specific engineering of TALEN. Using 5azadC with meganuclease allowed to stimulate the cutting frequency by nearly 20 fold in XPC fibroblasts and the engineered TALEN allowed a 40 fold-increase in frequency. With both strategies I obtained genetic correction events by repair matrix introduction in the targeted locus with a near 3% frequency. The characterization of corrected clones with the XPC TALEN shows genomic correction in the ninth exon, a restoration of the XPC protein expression and cell survival following UV exposure, thus demonstrating fully recovered normal repair activity by NER. This study represents the first evidence of genetic correction of XPC-deficient cells by a targeted approach.