Identification of Hox Genes Controlling Thrombopoiesis in Zebrafish Page: 2
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Sundaramoorthi, Hemalatha. Identification of hox genes controlling thrombopoiesis in
zebrafish. Doctor of Philosophy (Biochemistry and Molecular Biology), December 2015, 137
pp., 3 tables, 46 figures, chapter references.
Thrombocytes are functional equivalents of mammalian platelets and also possess
megakaryocyte features. It has been shown earlier that hox genes play a role in megakaryocyte
development. Our earlier microarray analysis showed five hox genes, hoxa]0b, hoxb2a, hoxc5a,
hoxclib and hoxd3a, were upregulated in zebrafish thrombocytes. However, there is no
comprehensive study of genome wide scan of all the hox genes playing a role in
megakaryopoiesis. I first measured the expression levels of each of these hox genes in young
and mature thrombocytes and observed that all the above hox genes except hoxclib were
expressed equally in both populations of thrombocytes. hoxclib was expressed only in young
thrombocytes and not in mature thrombocytes. The goals of my study were to comprehensively
knockdown hox genes and identify the specific hox genes involved in the development of
thrombocytes in zebrafish. However, the existing vivo-morpholino knockdown technology was
not capable of performing such genome-wide knockdowns. Therefore, I developed a novel cost-
effective knockdown method by designing an antisense oligonucleotides against the target
mRNA and piggybacking with standard control morpholino to silence the gene of interest. Also,
to perform knockdowns of the hox genes and test for the number of thrombocytes, the available
techniques were both cumbersome or required breeding and production of fish where
thrombocytes are GFP labeled. Therefore, I established a flow cytometry based method of
counting the number of thrombocytes. I used mepacrine to fluorescently label the blood cells and
used the white cell fraction. Standard antisense oligonucleotide designed to the central portion
of each of the target hox mRNAs, was piggybacked by a control morpholino and intravenously
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Sundaramoorthi, Hemalatha. Identification of Hox Genes Controlling Thrombopoiesis in Zebrafish, dissertation, December 2015; Denton, Texas. (https://digital.library.unt.edu/ark:/67531/metadc822768/m1/2/: accessed April 23, 2024), University of North Texas Libraries, UNT Digital Library, https://digital.library.unt.edu; .