Browsing by Subject "Transposon"

Now showing 1 - 3 of 3
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    Development of the larval zebrafish as a genetic model for the nicotine response.
    (2010-08) Petzold, Andrew Michael
    Tobacco use is predicted to result in over 1 billion deaths worldwide by the end of the 21st century. How genetic variation contributes to the observed differential predisposition in the human population to drug dependence is unknown. The zebrafish (Danio rerio) is an emerging vertebrate model system for understanding the genetics of behavior. We developed a nicotine behavioral assay in zebrafish and applied it in a forward genetic screen using gene-breaking transposon mutagenesis. We used this method to molecularly characterize bdav/ cct8 and hbog/gabbr1.2 as mutations with altered nicotine response. Each have a single human ortholog, identifying two points for potential scientific, diagnostic, and drug development for nicotine biology and cessation therapeutics. We show this insertional method generates mutant alleles that are reversible through Cre-mediated recombination, representing a conditional mutation system for the zebrafish. Additionally, we developed a conditioned place preference assay for use with larval zebrafish. This assay allows for the perturbation of the differences in genetic function between the physiological and learned response representing one of the first associative learning based assays in the larval zebrafish. The combination of this reporter-tagged insertional mutagen approach and zebrafish provides a powerful platform for a rich array of questions amenable to genetic-based scientific inquiry, including the basis of behavior, epigenetics, plasticity, stress, memory, and learning.
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    Development of tools for genome engineering in swine.
    (2009-09) Carlson, Daniel Fred
    Heightened interest in relevant models for human disease, in the production of transgenic livestock for biomedical applications, and new and pending releases of genome sequences for rat, cow, and pig have increased the need for improved methods for germline transgenesis. Transpositional transgenesis (TnT) offers an efficient and precise mechanism for genome integration of transgene DNA that avoids incorporation of CG-rich vector DNA and multi-copy transgene concatemerization that can lead to suppression of gene expression and transgene instability. We have developed and tested a transposon toolbox (including Sleeping Beauty, Tol2, piggyBac, and Passport) in pig cells. We have also demonstrated the activity of Cre and Flp recombinases in cultured pig cells and have implemented that technology for regulated activation of gene expression in swine. The application of transposon and recombinase technologies significantly enhances both the means and possible complexity of pig genetic modification. The use of enhanced cis and trans components of the Sleeping Beauty (SB) transposon system for animal transgenesis by pronuclear injection (PNI) resulted in tremendous improvement in the creation of transgenic laboratory mice, rats, and pig embryos, increasing both the frequency of transgenic founders, and the number of transgenes per founder, overall elevating the number of potential transgenic lines by 10-20-fold. Genetic modification of pigs by tandem pig transgenesis and cloning also benefits from TnT, resulting in multiple independent insertions per cellular clone which permits the assessment of several alleles upon segregation from a single founder. Finally, towards the development of a porcine model of cystic fibrosis (CF), we’ve created a mouse phenocopy of CF based on RNA interference using the SB transposon system. Building on insights gained in CFTRRNAi mouse, we’ve developed tightly regulated CFTRRNAi alleles in pigs to circumvent the lethal neonatal meconium ileus that confounds the CFTR-knockout model, hopefully simplifying investigation of postnatal CFTR deficiency in a large animal. Given its simplicity, versatility and high efficiency, TnT represents a compelling non-viral approach to modifying the porcine germline.
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    Insights into determinants of cancer susceptibility, initiation, and progression:studies on medulloblastoma and Histiocytic Sarcoma in mouse models
    (2012-12) Been, Raha Allaei
    This dissertation presents a discussion of both perigestational dietary influence on cancer predisposition as well as somatic genetic determinants of cancer development. Both projects used genetically engineered mouse models of cancer. The introductory chapter gives a brief historical introduction to cancer, background information on models of cancer, and a short description of our current understanding of cancer. Chapter two presents data on how maternal diet can affect the risk for medulloblastoma in offspring. Medulloblastoma presents a dismal prognosis even for the patients who are successfully treated. Prevention strategies are therefore of great interest in addressing this disease. Chapter three discusses a mouse model of Histiocytic Sarcoma (HS) that was developed to identify genes that can contribute to initiation and cause progression of disease. The genetics of HS are not well understood. Our model could provide important information on molecular targets that can be used to treat this dreadful disease. The final, fourth chapter, provides a brief and broad overview of some of the major future likely sources of cancer control success with a focus on new research.