Using RNAi technology to down-regulate Six2 expression in vitro
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2008
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The Brachyrrhine (Br) mutant mouse, which displays frontonasal dysplasia and renal hypoplasia, has previously been described. Linkage analysis mapped the semi-dominant Br mutation close to the homeobox transcription factor Six2, which is normally expressed in the facial and metanephric mesenchyme during embryonic development. The purpose of this study is to evaluate the role of Six2 in craniofacial and renal branching morphogenesis by quantifying its expression level in the facial prominences and using immunohistochemistry to visualize branching of the ureteric bud in Br mice. In addition, an in vitro system utilizing RNA interference (RNAi) technology was developed to determine whether Six2 could be experimentally down-regulated. Medial nasal (MNP), lateral nasal (LNP) and maxillary prominences (MAX) of EII.5 embryos were dissected and Six2 expression was measured with qRT-PCR. Six2 expression was highest in the MNP, with about 2-fold less in the MAX, and 3-fold less in the LNP of wild-type embryos. Our data indicate a haploinsufficient pattern of Six2 expression in each of the three sets of facial prominences. In addition, kidney organ explants were dissected from E13.5 mouse embryos and immunostained to reveal differences in ureteric bud branching patterns between the three genotypes (+1+, Br/+ and Br/Br). We confirmed a down-regulation of Six2 in a cell culture system utilizing five RNAi constructs. These data indicate a lack of Six2 expression may playa role in the development of a median facial cleft and its reduced effect in the kidney may lead to renal hypoplasia Additionally, an in vitro system was established that will allow experimental down-regulation of Six2 to assess effects on morphogenesis.
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Thesis (M.S.)--University of Hawaii at Manoa, 2008.
Includes bibliographical references (leaves 36-42).
vii, 42 leaves, bound 29 cm
Includes bibliographical references (leaves 36-42).
vii, 42 leaves, bound 29 cm
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Theses for the degree of Master of Science (University of Hawaii at Manoa). Biomedical Sciences (Physiology); no. 4278
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