Browsing Theses and Dissertations by Subjects
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Artificial chromosome transgenesis reveals long-distance negative regulation of rag1 in zebrafishDespite the essential roles played by the recombination activating genes (rag 1 and rag2) during V(D)J recombination, the mechanisms that restrict their expression to lymphoid cells are undefined. Using a novel approach to achieve artificial chromosome transgenesis in zebrafish, we demonstrate that distal regulatory elements are critical to suppress rag 1 expression in inappropriate tissues. In contrast to smaller reporter gene constructs, 125 and 75 kb artificial chromosomes containing the zebrafish rag genomic locus directed GFP expression in a pattern reflective of endogenous rag 1. Mapping experiments identified a positive element 5' of rag 1 that enhances GFP expression in both lymphoid and non-lymphoid tissues and a negative element 5' of rag 1 that specifically suppresses GFP expression in the skeletal muscle. Our transgenic zebrafish also express GFP in olfactory neurons which we show represent an authentic rag] expression site in zebrafish.
Characterization of zebrafish mutant merlot as a non-mammalian vertebrate model for congenital anemia due to protein 4.1 deficiencyThe zebrafish mutant merlot (mot) is characterized by onset of a severe anemia at 96 hours post fertilization. We performed whole mount RNA in situ hybridization and showed that ihe process of primitive erythropoiesis is not interrupted in the mot embryos. Blood analysis demonstrated that mot suffers from a severe congenital hemolytic anemia. Using the TUNEL assay, we detected apoptotic erythroid progenitors in the kidneys. We performed electron microscopic analysis and detected membrane abnormalities and a loss of the cortical membrane organization in the mot cells. We used positional cloning techniques with a candidate gene approach to demonstrate that mot encodes the erythroid specific isoform of protein 4.1R, a critical component of the red blood cell membrane skeleton. Sequence analysis of 4.1R eDNA detected nonsense point mutations in both alleles of mot resulting in premature stop codons. We performed linkage analysis and transgenic rescue experiments to provide further confirmation that the molecular defect in the protein 4.1R is the underlying cause of anemic phenotype in mot fish. This study presents the zebrafish mutant merlot as the first characterized non-mammalian vertebrate model of congenital anemia due to a defect in protein 4.1R integrity.