Browsing by Subject "Sleeping Beauty"

Now showing 1 - 3 of 3
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    Cancer gene discovery using somatic transposon mutagenesis in the mouse and systems for validation of identified candidate cancer genes and pathways.
    (2012-07) Moriarity, Branden Scott
    The conditional Sleeping Beauty (SB) transposon mutagenesis system has proven to be a successful method for cancer gene identification in solid tumors. Using tissue specific Cre recombinases (TSP-Cre) to selectively activate SB mediated mutagenesis in tissues of interest has yielded numerous novel candidate cancer genes in leukemia/lymphoma, colorectal, liver, and pancreatic cancers. We implemented the SB system for the identification of genes involved in osteosarcoma (OS). OS is the most common cancer of the bone and third most common cancer in children and adolescents. The 5-year survival rate of OS patients is 60-70%, though with metastatic disease it drops to less than 20%-30%. Identification of genes responsible for OS development and metastasis has been difficult due to its genomic instability and subsequent complexity. Consequently, there is a pressing need to identify the genes and pathways governing OS development and metastasis. To this end, we performed a forward genetic screen utilizing the conditional Sleeping Beauty (SB) transposase and mutagenic T2/Onc transposon system in Osx-Cre mice on a predisposing Trp53LSL-R270H/+ or wild type background to induce random somatic mutations that induce OS development and metastasis. One hundred and nineteen OSs were isolated from 96 predisposed and 20 wild type mice undergoing SB mutagenesis, with a subset of animals harboring metastases to the liver and/or lungs. Over 100 candidate cancer genes, both known and novel, were identified by analysis of recurrent T2/Onc insertion sites. In order to validate the candidate cancer genes identified in our screen, we developed a recombinase-based system for efficient assembly of vectors to over express or knock down 1-6 genes in mammalian cells. Further, these vectors were constructed in Piggybac transposons to allow for stable and reversible integration of the vector via transposition. In addition to cDNA over expression or shRNA knock down, we also developed methods for generation and selection of candidate cancer gene knock out cell lines using TAL effector nucleases (TALENs).
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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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    The Sleeping Beauty Transposon System For Forward And Reverse Genetic Studies Of Liver Cancer
    (2017-04) Tschida, Barbara
    Hepatocellular carcinoma (HCC) is the second leading cause of death from cancer globally and increasing in prevalence. HCC usually occurs in the context of chronic liver damage. The specific genetic alterations promoting HCC have been difficult to identify due to the genetic heterogeneity of HCC and the prevalence of large chromosome aberrations. To study the pathogenesis of HCC, we used Sleeping Beauty (SB) transposons for both a forward genetic screen and reverse genetic studies of genes identified in HCC mouse models, altered in human HCC, or components of the Hepatitis B virus, a major HCC risk factor. Hepatic steatosis is a common chronic liver disease linked to HCC development. We used conditional SB transposon insertional mutagenesis in mice with diet and ethanol induced hepatic steatosis to model steatosis-associated HCC and perform a forward genetic screen for molecular drivers. We compared the results from this screen to human HCC data from multiple sources. We found an increase in HCC in females in both mice and humans with hepatic steatosis, reducing the typical male sex bias of HCC. We identified over 200 genes candidate steatosis-associated HCC genes in mice, many of which are altered in human steatosis and alcohol associated HCC. We identified an association between protein kinase A/cyclic AMP signaling pathway alterations and steatosis-associated HCC, and NAT10 overexpression and HCC, both of which could potentially be targeted therapeutically. We used SB transposon-based gene delivery for reverse genetic studies testing candidate oncogenes identified in human HCC, complementary mouse models including the steatosis-associated HCC screen, and Hepatitis B viral genes for their roles in promoting liver tumorigenesis. These studies revealed new oncogenic roles for genes tested in normal livers, in mice of both sexes, in steatotic livers, and in fibrotic livers. We generated several new mouse models of HCC that could be used for mechanistic studies or therapeutic testing.