SOX4 基因轉殖斑馬魚的造血系統研究

Hematopoietic study of SOX4 transgenic zebrafish

謝孟珊(Meng-Shan Hsieh)

指導教授 ： 林亮音

國立臺灣大學/醫學院/醫學檢驗暨生物技術學研究所/碩士(2015年)

https://doi.org/10.6342/NTU.2015.10726

摘要

無資料

關鍵字

SOX4 ； 基因轉殖斑馬魚 ； Tol2轉位子 ； 造血 ； 骨髓惡性血液疾病

並列摘要

In the present study, taking advantages of rapid growth and transparent embryo, together with the similarity of gene sequence and hematopoiesis to mammals, zebrafish has been a good animal model to study on the development of hematopoiesis and hematologic malignancies. SOX4 (SRY sex determining region Y-box 4) is a transcription factor belonging to SOX family. Overexpression of SOX4 has been found in a variety of cancers. It is indicated that desregulated expression of SOX4 was associated with leukemogenesis in acute myeloid leukemia (AML) patients with CEBPA mutation. Therefore, SOX4 or its downstream molecules might be potential therapeutic targets for treating this subtype of AML patients. Furthermore, SOX4 has also been found to be strongly related to cell proliferation, survival, and differentiation; and overexpression of SOX4 in myeloid progenitor cells resulted in a blockage to myeloid maturation. Although these findings have provided preliminary concepts, further study is required to understand the mechanism and the impact of SOX4 on hematopoiesis or myeloid malignancies. Thus, the aim of this study is to establish a transgenic zebrafish model expressing human SOX4 under the control of myeloid specific promoter, and to explore the role of SOX4 on zebrafish hematopoiesis from embryo to adult stages.
Oncomine research analysis first revealed that the expression of SOX4 is relatively increased in patients with AML compared with normal controls (p<0.0001). In addition, we confirmed that the HMG domain and TAD domain of SOX4 gene were conserved between species by phylogenetics and sequence alignment. By using Multisite Gateway system○R and Tol2 transposon technology, we established an expression vector including spi1 promoter, human SOX4 gene and EGFP. This constructed vector was then injected into zebrafish embryos. After a serious of fluorescent selection, stable transgenic SOX4 zebrafish were generated. Quantitative real-time polymerase chain reaction (qRT-PCR) revealed that SOX4 could be detected at 20 hpf (TG1, 0.2x103 copies; TG2, 1.7x103 copies), and then increased gradually during development. At 5 dpf, level of SOX4 in TG1 and TG2 was up to 2x103 copies and 10x103 copies, respectively. However, there were no significant differences in the expression of hematopoiesis-related transcription factors, in spite of the expression of SOX4. We also found that there were no significant difference of myeloperoxidase (mpo) expression in transgenic zebrafish TG1 and TG2, compared with control fish by using whole-mount in situ hybridization (WISH) technique with p value of 0.11 and 0.89, respectively. In addition, Tg(spi1:SOX4-EGFP/cd41:EGFP) demonstrated normal development of hematopoietic stem cells; and Tg(spi1:SOX4-EGFP) zebrafish had normal granulopoiesis of myeloid cells. These results indicated that the SOX4 transgenic zebrafish had normal hematopoiesis in the larval stage.
Regarding to the adult stage, 5 and 9-month-old SOX4 transgenic zebrafish were examined. At age of 5 months, out of 6 transgenic zebrafish, 4 were found to have increased myelopoiesis (M/E=2.20 to 6.42 vs. 1.70) in kidney marrow, especially immature myeloid cells; but showing normal pattern in peripheral blood (PB). At age of 9 months, out of 6 transgenic zebrafish, more increased myelopoiesis (M/E=4.88 to 9.09 vs. 3.31) and immature cells were found in kidney marrow. Intriguingly, some immature cells were found in PB smear at age of 9 months.
Taken together, we proposed that continuous overexpression of SOX4 in myeloid cells would lead to an expansion of myeloid progenitors and differentiation arrest in kidney marrow as well as abnormal myeloid cells in PB from SOX4 transgenic zebrafish at adult stage. These defects resemble the blood pattern of myeloid malignancy in human beings.

並列關鍵字

SOX4 ； transgenic zebrafish ； Tol2 transposon ； hematopoiesis ； myeloid malignancy

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