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DNA double strand breaks (DSBs) are primarily repaired in eukaryotic cells by two different mechanisms – non-homologous end joining (NHEJ) or homologous recombination (HR). In mammalian somatic cells the balance between the two highly favours NHEJ. Gene targeting is a technique that exploits HR repair to alter a defined gene locus. While it holds potential to be implemented as a treatment option for several diseases, the outlook for using it in a clinical setting has been obstructed by a low gene targeting efficiency. This has been coupled to the low frequency of HR in mammalian cells. With the intention of shifting the repair balance, antibodies against DSB repair proteins will be introduced into mammalian cells. It is predicted that by targeting key repair proteins with antibodies, a compensatory increase in the frequency of HR can be fostered, ultimately resulting in improved gene targeting. / xv, 168 leaves : ill. ; 29 cm

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Additional advisors: Ada Elgavish, Vithal Ghanta, Hui-Chen Hsu, Thane Wibbels. Description based on contents viewed June 11, 2008; title from title screen. Includes bibliographical references.

This thesis describes the establishment of a precise gene targeting methodology in the silkworm Bombyx mori by technologies based on engineered endonucleases. Two classes of engineered endonucleases, ZFNs and full length TALENs were used for creating DSBs at specified sites in the colour marker genes (BmBlos2 and Bmwh3). Direct embryo microinjection of engineered nucleases mRNA were performed and let the nuclease proteins to disrupt the functions of these marker genes by creating DSBs and inducing error prone NHEJ mechanism. These experiments showed that both ZFNs and TALENs could be used for targeted gene disruption in silkworms.

The ability to precisely modify the mouse genome is an invaluable tool for any researcher. If an artificial epitope sequence is integrated into target loci in specific cell types, it is possible to generate mice with these cells specifically tagged with the epitope, which can be used for many subsequent studies. Homologous recombination and the Cre/loxP system have been used to generate targeted and conditional transgenic mice, which have provided the basis for many studies into gene function. However, in recent years, improvements in technology have led to the development of RNA and protein based methods of specifically editing DNA sequences at user-selected loci. This thesis aimed to investigate the effectiveness of the novel gene targeting methods TALEN and CRISPR/Cas9. It also aimed to utilize different strains of mice generated using the Cre/loxP system in Trichuris muris, an animal infection model of the human disease Trichuris trichiura. TALENs use a pair of protein-based monomers specific to the sense and anti-sense strand of a target DNA sequence to dimerise a FokI nuclease and initiate a deletion in the genome. As a study into the practical use of this emerging technology TALENs were generated to target Oct-4 (a stem cell marker) in order to integrate an artificial epitope sequence, which could be used for enrichment experiments. The CRISPR/Cas9 is a very efficient RNA-based system used for modifying a target sequence. This system has been utilized to integrate an epitope sequence into the Rosa26 locus, downstream of a floxed STOP codon. This allows for expression of the epitope following the introduction of tissue restricted Cre recombinase. IL-1 is an important cytokine in the immune response towards T.muris. IL-1R1 was conditionally removed in CD4 cells and the role of IL-1 signaling in developing Th1, Th2 and Th17 responses was then studied. Interestingly, IL-1R1fl/fl CD4Cre mice could generate Th1 and Th2 response but showed a reduction in IL-22 and IL-17 production, two key Th17 cytokines. Infected IL-1R1fl/fl CD4Cre mice also displayed increased gut morphology and goblet cell hyperplasia. Therefore, it was concluded that IL-1 signaling from CD4 cells has an important role in host defense and the development of a full Th17 response. It was also shown that removing IL-1R1 in naïve mice had no affect on lymphocyte development. IL-10 is an anti-inflammatory cytokine expressed by gut macrophages, which contributes to homeostatic control of the immune system. IL-10R was specifically removed in the macrophage specific Cre lines LysMCre and also in CX3CR1Cre as a way of comparing the two Cre drivers. The mice were then infected with T.muris and displayed significant inflammation and also failure to expel the worms in the LysMCre model. This suggests a role for this model in future studies of gut macrophages. Clearly, animal models are very important in the study of gene function and also as a method of assessing the application of new technologies such as CRISPR/Cas9. Future work with the artificial epitope specifically targeted into important cell lines will form the basis of many important studies directly applicable to human disorders. As the technologies improve, the scope for developing therapeutics increases and genetic modification has an immeasurable role to play.

Commonly, when it is desirable to replace an essential gene with an allelic series of mutated genes, or genes with altered expression patterns, the complementing constructs are introduced into heterozygous plants, followed by the selection of homozygous null segregants. To overcome this laborious and time-consuming step, the newly developed two-component system utilizes a site-specific recombinase to excise a wild-type copy of the gene of interest from transformed tissues. In the first component (the first vector), a wild-type version of the gene is placed between target sequences recognized by FLP recombinase from the yeast 2 μm plasmid. This construct is transformed into a plant heterozygous for a null mutation at the endogenous locus, and progeny plants carrying the excisable complementing gene and segregating homozygous knockout at the endogenous locus are selected. The second component (the second vector) carries the experimental gene along with the FLP gene. When this construct is introduced, FLP recombinase excises the complementing gene, leaving the experimental gene as the only functional copy. The FLP gene is driven by an egg apparatus specific enhancer (EASE) to ensure excision of the complementing cDNA in the egg cell and zygote following floral-dip transformation. The utility of this system is being tested using various experimental derivatives of the essential sucrose-proton symporter, AtSUC2, which is required for photoassimilate transport.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22854号 / 農博第2437号 / 新制||農||1082(附属図書館) / 学位論文||R2||N5314(農学部図書室) / 京都大学大学院農学研究科地域環境科学専攻 / (主査)教授 本田 与一, 教授 田中 千尋, 准教授 坂本 正弘 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Because of shared physiological, anatomical and metabolical features with humans, mice have served for a long time as mammalian disease models. In particular, these last ten years have been the golden age for this favoured model animal. Human and mouse genome projects show that there is 95% genome homology. Spurred by this fact, research attention has shifted from reading these sequences to deciphering the functions of these genes. The 1980s saw the remarkable achievement of homologous recombination in mammalian cell culture systems. Later in the 1990s, innovative gene trapping strategies were developed to enabled random mutagenesis. Today, the goal is to generate more versatile tools to avoid limitations posed by these earlier mutagenesis strategies. Many public and private research centers have united with the aim of mutating all mouse genes. In order to achieve this mutagenesis, the first requirement is a set of practical and efficient viral or plasmid based vectors that can be used globally in the genome. This will be aided by advances in understanding of biological events such as gene transcription, recombination, and embryonic stem cell cycle. In addition, technical improvements such as vector development, precise cell culture assay, and recombinant DNA delivery will also be important. The vector design work in this PhD thesis encompasses 0.00001 % ofthese efforts but may to out to be highly relevant...

To understand the physiological function of glutaredoxin, a thiotransferase catalyzing the reduction of mixed disulfides of protein and glutathione, we generated a line of knockout mice deficient in the cytosolic glutaredoxin 1 (Grx1). To our surprise, mice deficient in Grx1 were not more susceptible to acute oxidative insults in models of heart and lung injury induced by ischemia/reperfusion and hyperoxia, respectively, suggesting that either changes in S-glutathionylation status of cytosolic proteins are not the major cause of such tissue injury or developmental adaptation in the Glrx1-knockout animals alters the response to oxidative insult. In contrast, mouse embryonic fibroblasts (MEFs) isolated from Grx1-deficient mice displayed an increased vulnerability to diquat and paraquat, but they were not more susceptible to cell death induced by hydrogen peroxide (H2O2) and diamide. A deficiency in Grx1 also sensitized MEFs to protein S-glutathionylation in response to H2O2 treatment and retarded deglutathionylation of the S-glutathionylated proteins, especially for a single prominent protein band. Additional experiments showed that MEFs lacking Grx1 were more tolerant to apoptosis induced by tumor necrosis factor αplus actinomycin D. These findings suggest that various oxidants may damage the cells via distinct mechanisms in which the action of Grx1 may or may not be protective and Grx1 may exert its function on specific target proteins.