[問題] 病毒論文的實驗

看板Biotech (生命科學)作者paua (waiting for good news)時間19年前 (2006/04/15 18:05)推噓2(2推 0噓 0→)

留言2則, 2人參與討論串1/3 (看更多)

我在看這篇 Herpes simplex virus disrupts NF-κB regulation by blocking its recruitment on the IκBα promoter and directing the factor on viral genes. 出處: THE JOURNAL OF BIOLOGICAL CHEMISTRY 2006, 281(11): 7110–7117 作者: Carla Amici, Antonio Rossi, Antonio Costanzo, Stefania Ciafre, Barbara Marinari, Mirna Balsamo, Massimo Levrero, and M. Gabriella Santoro (不好意思啊, 為了打字方便, 直接把kappa--> k; alpha--> a) 這篇論文主要探討病毒感染後, 細胞NF-kB上升過程中可能的原因. 論文前幾個實驗已經推論幾個時間點: 病毒感染--> IKK活性上升(感染後零小時及第三個小時)--> IkBa降解, 且NF-kB在感染後期不會被recruit到IkBa promoter(使IkBa產生, 回饋抑制NF-kB活性), 而會被recruit到病毒基因(ICP0)的promoter. 問題來了, 實驗中將ICP0 promoter放到實驗細胞株(HaCaT cell)中, 想要知道NF-kB被recruit到viral或cellular DNA, 可是我看了好久都看不懂. 原本我以為實驗是要看病毒基因是否有嵌入宿主細胞的基因裡, 但是老師說, 似乎這種病毒(HSV-1)不會嵌入宿主細胞.. 我不曉得這個實驗的目的為何, 麻煩請病毒達人指點指點一下, 謝謝 :) 在此附上實驗的原文: Viral ICP0 Promoter Shows Remarkable Avidity for NF-kB To investigate whether the differences observed in NF-kB recruitment could be a consequence of the different status of viral and cellular DNA organization (episomal versus chromosomal), we have generated HaCaT cells (HaCaT-ICP0-Luc cells) in which the ICP0 promoter controlling the expression of the luciferase reporter gene is stably integrated into the chromatin structure. HaCaT-ICP0-Luc cells were infected with HSV-1, and, at different times post-infection, were processed for luciferase or ChIP analysis. As shown in Fig. 5B, HSV-1 infection induces luciferase activity in these cells, indicating that the ICP0-Luc promoter is transcriptionally activated by the virus. In the same experiment, NF-kB recruitment to the integrated and to the free viral ICP0 promoters was analyzed at the end of the virus adsorption period and at 5 h post-infection, which correspond to the first and second waves of NF-kB activation, respectively. To discriminate the integrated form of the ICP0 promoter from the non-integrated viral promoter in the ChIP analysis, we have utilized the same upstream primer but different downstream primers (Fig. 5A). As shown in Fig. 5C, at the end of the 1-h adsorption period, NF-B is recruited to the IB promoter and to both forms (viral and integrated form) of the ICP0 promoter, indicating that during the first wave of NF-kB activation all the promoters analyzed behave similarly in respect to NF-kB recruitment. As expected, at 5 h p.i. NF-kB was not recruited to the IB promoter. Interestingly, at this time, NF-kB was recruited selectively to the viral form of the ICP0 promoter. When integrated into the host chromatin structure, the ICP0 promoter looses its ability to recruit the nuclear factor, behaving like IB (Fig. 5C). These results suggest that the differences in NF-kB recruitment observed between ICP0 and IB promoters could be due to differences in the status of DNA and/or to general chromatin modifications induced by HSV-1 during infection. -- ※ 發信站: 批踢踢實業坊(ptt.cc) ◆ From: 140.129.60.111