單核球細胞報導基因檢測系統的建立與應用

Establishment and Application of a Reporter Gene Assay System in Monocytes

劉嘉恩(Jia-En Liu)

指導教授 ： 顏伯勳

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/碩士(2022年)

https://doi.org/10.6342/NTU202200291

摘要

報導基因是一組具有易於觀察、測量和量化之表現型的基因。它們應該是高度靈敏、方便和可靠的，並能快速檢測到結果。報導基因通常會連接在調控基因的啟動子附近，替換外顯子，並測量未知 DNA 序列調控轉錄的能力。內含報導基因的質體一般以轉染的方式送入細胞內。然而，免疫細胞只能透過病毒轉導方式獲得質體。由於病毒感染免疫細胞的困難，沒有太多關於使用免疫細胞進行報導基因檢測的研究。在我們之前的研究中，我們建立了一個螢光素酶報導基因檢測系統，成功地測量了 HEK293T 細胞中 NF-kB 啟動子驅動的轉錄活性；然而，相同的系統在 CCL2 啟動子上無法作用。我們相信對趨化素啟動子的調節作用在免疫細胞中會更加明顯。因此，本研究在單核球細胞中建立了功能性報導基因檢測系統。使用慢病毒載體，將編碼紅色螢光蛋白和帶有NF-kB啟動子的螢光素酶之基因轉導到THP-1單核球細胞中，並進一步培養出表達紅色螢光蛋白的轉導細胞。用 TNFa 和 IFNr 處理成功地誘導了 NF-kB 啟動子驅動的螢光素酶報導基因的表現，並且在doxycycline誘導的 SP110b 表現後，該報導基因的表現增加了約 17 倍。該結果證明本研究創建的報導基因檢測系統可用於單核球細胞，也可用於其他細胞。此外，在載體中插入多個限制酶切位點的接頭序列有助於將各種啟動子替換到載體中，使其成為測試單核球細胞或其他細胞群中目標啟動子活性非常有用的平台。

關鍵字

報導基因 ； 單核球細胞 ； THP-1 ； 螢光素酶 ； NF-kB

並列摘要

Reporter genes are a herd of genes with phenotypes that are easy to observe, measure and quantify. They should be highly sensitive, convenient and reliable with a quickly detected result. Reporter genes would typically be attached to the regulatory region, replace exon, and measure the regulatory potential of an unknown DNA-sequence. Plasmids are usually integrated into cells through transfections. However, immune cells can only obtain exogenous DNA through transductions. There is not much data on conducting reporter gene assay using immune cells due to the difficulties of immune cells by viral infection. In our previous study, we established a luciferase reporter gene assay system to successfully measure the NF-kB promoter-driven transcription activity in HEK293T cells; however, the same system did not work on the CCL2 promoter. We believe that the regulatory effect on chemokine promoters would be more evident in immune cells. Therefore, in this study, a functional reporter gene assay system was established in monocytes. Using a lentiviral vector, the genes for encoding the red fluorescent protein and the luciferase with the NF-kB promoter were transduced into THP-1 monocyte cells, and the transduced cells that expressed red fluorescent protein were further sub-cloned. The treatments with TNFa and IFNr successfully induced the expression of the NF-kB promoter-driven luciferase reporter gene, and the expression was up-regulated up to 17 folds upon doxycycline-induced SP110b expression. This result proves that the reporter gene assay system created in this study can be used in monocytes and may also be used in other cells. In addition, a linker sequence with multiple cloning sites inserted in the vector facilitates various promoter replacement into the vector, making it a very useful platform for testing a target promoter activity in monocytes or other cell populations.

並列關鍵字

reporter gene ； monocyte ； THP-1 ； luciferase ； NF-kB

參考文獻

Aasland, R., Gibson, T.J., and Stewart, A.F. (1995). The PHD finger: implications for chromatin-mediated transcriptional regulation. Trends Biochem Sci 20, 56-59. 10.1016/s0968-0004(00)88957-4.

Alam, J., and Cook, J.L. (1990). Reporter genes: application to the study of mammalian gene transcription. Anal Biochem 188, 245-254. 10.1016/0003-2697(90)90601-5.

Bendall, L. (2005). Chemokines and their receptors in disease. Histol Histopathol 20, 907-926. 10.14670/hh-20.907.

Bloch, D.B., Nakajima, A., Gulick, T., Chiche, J.D., Orth, D., de La Monte, S.M., and Bloch, K.D. (2000). Sp110 localizes to the PML-Sp100 nuclear body and may function as a nuclear hormone receptor transcriptional coactivator. Mol Cell Biol 20, 6138-6146. 10.1128/mcb.20.16.6138-6146.2000.

Chalfie, M., Tu, Y., Euskirchen, G., Ward, W.W., and Prasher, D.C. (1994). Green fluorescent protein as a marker for gene expression. Science 263, 802-805. 10.1126/science.8303295.