M.S. - Cell and Molecular Biology
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ItemInsertion of a functional copy of six2 to generate a transgenic mouse via piggybac transposase([Honolulu] : [University of Hawaii at Manoa], [August 2013], 2013-08)Identification of gene function based on known mutations remains an integral objective in the field of basic science. The adult Br heterozygous mouse displays a reduced number of nephrons in association with chronic renal failure, hypertension and reduced Six2 expression during embryonic renal morphogenesis. The purpose of this study was to determine whether a functional copy of Six2 could be inserted into the Br mouse genome in an effort to overexpress Six2. After processing and isolation by restriction enzyme digestion and pulse field gel electrophoresis, 26kb Six2 BAC DNA fragment was then cloned into the mGENIE-3-BAC transposon vector via the in vitro Gibson Assembly method. The mGENIE-3-BAC-Six2 construct was subsequently confirmed by colony PCR, restriction enzyme digestion; the construct was also tested for functionality and expression in human cell lines and transgenic mice. Our data indicates that our single plasmid transposase-mediated approach to transgenesis requires fewer embryos, while capable of incorporating 42kb of exogenous DNA into the mouse genome.
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ItemNovel insights on the role of selenoprotein p in sperm viability([Honolulu] : [University of Hawaii at Manoa], [August 2012], 2012-08)Selenium (Se) is a micronutrient essential for life in many organisms. Selenium is incorporated into selenoproteins as the twenty-first amino acid, selenocysteine (Sec), and has antioxidant properties. One member of the family of twenty-five selenoproteins in humans is Selenoprotein P (Sepp1). This protein is synthesized primarily in liver and is proposed to transport selenium throughout the body, particularly to the brain and testes. Sepp1 knockout (KO) mice on (normal) diets without selenium supplementation have decreased selenoprotein expression in brain and testes. Previous studies have suggested that Sepp1 male KO mice are infertile due to kinks in the flagellum of spermatozoa, greatly reducing sperm motility, therefore leading to dramatically decreased fertility. In this two-part study, our first objective was to further understand the role of Sepp1 on sperm viability. We hypothesize that Sepp1 plays a critical role in sperm DNA viability independent of motility, potentially through modulating glutathione peroxidase 4 (GPx4) biosynthesis. GPx4 is another selenoprotein known to protect cells from membrane lipid peroxidation and has been implicated in development and fertility [71]. The second objective of this study was to introduce a novel application to rescue Sepp1 global expression in knockout animals (Sepp1r/r CMV+) using Cre recombinase transgenic mice. We addressed the role of Sepp1 in sperm DNA viability with intracytoplasmic spermatozoa injections (ICSI) of Sepp1 KO sperm into wild type oocytes. Surrogate female mice carrying embryos resulting from injection of Sepp1-/-sperm resulted in a 72.3% reduction in live pups born compared to Sepp1 heterozygous control sperm. Our results from the ICSI experiments, in which sperm were directly injected into oocytes without flagella, suggest that Sepp1 is critical for sperm DNA viability independent of motility. We show through western blot analysis that GPx4 levels are significantly decreased in the testes and epididymides of Sepp1 KO mice, whereas Sepp1r/r CMV+ rescue mice had restored expression levels comparable to the Sepp1 wild type (Sepp1 WT). Immunohistochemistry studies using an antibody against GPx4 further confirmed that GPx4 levels were undetectable in fresh Sepp1 KO mouse sperm, while GPx4 levels in Sepp1r/r CMV+ rescue mice were similar to those of wild type controls. Cre-Lox recombination is a commonly used genetic tool for site-specific gene deletion. However, we demonstrate that this system can be used to rescue gene expression as well, restoring the expression of Sepp1 in KO mice. We show that this approach produced viable progeny of the systemic Sepp1r/r CMV+ (rescue) mice that express the CMV-Cre driven Sepp1 gene in all tissues. We confirmed through the Morris Water Maze (MWM) and other behavior assays that in contrast to Sepp1 KO mice, Sepp1r/r CMV+ mice had normal neuromotor function and memory compared to Sepp1+/+. Successful implementation of this method can further be utilized to restrict gene expression to specific cells. Our study presents new data showing that Sepp1 is crucial for viability of sperm DNA, potentially through regulation of GPx4 levels. Furthermore, we demonstrate an innovative method for restoration of gene expression using the Cre recombinase transgenic system, which can be applied to restrict gene expression to specific cells.
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ItemInvestigation of selenoprotein k function and associated proteins([Honolulu] : [University of Hawaii at Manoa], [May 2012], 2012-05)Selenoprotein K (SelK) is a small (16kD) single pass transmembrane protein localized to the ER. An Src Homology 3 (SH3)-binding domain within the amino acid sequence was exploited to identify potential protein-protein interactions. The effects of SelK deficiency in immune cells and effector calcium flux served as a model system to elucidate a function for SelK. Immunoprecipitation and mass spectrophotometry identification of potential binding partners returned Arf-GAP2 and pyruvate kinase as possible binding partners. Plasmid DNA vectors were designed for use in the Two-Hybrid system to confirm preliminary data of suspected interactions with the creation of fusion proteins coding for Vav1 or Vav2 SH3 domain and the SelK SH3 binding domain. Protein-protein interaction could not be confirmed in this system however SelK seems to play a role in receptor mediated calcium flux, and subsequent low-level production of nitric oxide by way of neuronal nitric oxide synthase.