LabMeeting-小嬿
由 EVKVLIN 在 二, 03/12/2013 – 00:00 發表 LIN Lab 2016 LabMeeting mmiiee 小嬿
2008/08/27
小女子深受luciferase assay的internal control困擾
所以上網找其他人有沒有這類的東西
看到了一個網址
http://www.protocol-online.org/biology-forums/posts/11321.html
可是這個人的問題是他的internal control會降低
跟我的不太一樣
後面有個人(doles)有建議 可以試試看phRL vector
是個合成的RL, 據說比較不會受到non specific的activator 影響
而且我剛剛又去看了一下珮雯學姊的論文
他用的就是Promega的phRL-TK
是因為這個原因所以他做起來才不會受影響嗎?
請老闆大人提示~~~~
謝謝
所以上網找其他人有沒有這類的東西
看到了一個網址
http://www.protocol-online.org/biology-forums/posts/11321.html
可是這個人的問題是他的internal control會降低
跟我的不太一樣
後面有個人(doles)有建議 可以試試看phRL vector
是個合成的RL, 據說比較不會受到non specific的activator 影響
而且我剛剛又去看了一下珮雯學姊的論文
他用的就是Promega的phRL-TK
是因為這個原因所以他做起來才不會受影響嗎?
請老闆大人提示~~~~
謝謝
2008/08/29
送MGM定序SOP
DNA 0.5-0.6 ug
Primer 4 ul of 2 uM
H2O to 8 ul
第一次做不出來
若想不到明顯錯誤處
可再送一次
若想不到明顯錯誤處
可再送一次
2008/09/09 Lab Meeting 心得彙整
小嬿: 講得很清楚喔!知道已給妳太多paper了讀不完(sorry) 但是,可否,拜託,先讀一下這四頁知識,幫我們summarize 一下. 我覺得瞭解這篇以後,對往後妳和小丸子的實驗設計非常有用(才不會走錯路)因為這實在是太顛覆我過去的想法了!
2008/09/23 Lab Meeting 心得彙整
小嬿: (1)BC- 1及P3HR1的fuctional assay真的很有趣. 看來gZ及cZ都有function.tZ大概真的是一個Z的suppressor. (2)請密切注意Ingrid及小丸子的ratio測試結果,很有可能DLR中activator及reporter比一直以來都把妳教錯了! Sorry.
小嬿: (1)BC- 1及P3HR1的fuctional assay真的很有趣. 看來gZ及cZ都有function.tZ大概真的是一個Z的suppressor. (2)請密切注意Ingrid及小丸子的ratio測試結果,很有可能DLR中activator及reporter比一直以來都把妳教錯了! Sorry.
2008/10/07 Lab Meeting 心得彙整
小嬿:(1) 多張slide空白處大於二分之一, 宜將內容放大.(2)promoter和transcription initiation site(箭頭處)要分清楚,尤其在解釋第一個實驗結果時,要將它們指出來,順便把三種status(Pol II stalls near the transcription start site, locates throughout the genes, and no Pol II)的分類帶出來.(3)最後作者是想做二個不互相抵觸的推論(A)Pol II pausing is for transcription repression(現象一,換成另一種mutantToll-rm9/10,那些在Toll-10b被抑制的基因就開始表現了,而它們原先Pol II pausing的狀態也都變成Pol II均勻分佈的狀態.現象二,snail是已知在Toll-10b中持續表現的一個repressor,作者發現接近60%被snail represses的基因也有Pol II stalling狀態)(B)Pol II pausing prepares genes for later expression (e.g. rapidly induction for hsp70, next-stage developmental genes including heart and muscle in the Toll-10b etc).(4)萬一這學期分數沒有上次高,都是我的錯,選了一篇太艱深的paper了…(先行懺悔去)
2008/10/14 Lab Meeting 心得彙整
小嬿:EBV gZ DLR 實驗條件決定(activator:BHLF1-Luc(1:4), SV40-RL as internal control);請繼續titrate EBV R/DN1-Luc條件(internal control 為？);請幫忙歸納兩組結果(一)gZ等clone送入EBV+ve細胞中叫不出endogenous Z的Western blot analysis、(二)TPA及SB對PELs disrupt latency 的效果(請加入PWW那一張以及這篇的圖一). 下週meeting就報這二個結果.Thanks.
2008/10/21 Lab Meeting 心得彙整
小嬿: TPA對EBV的Raji, B95-8, KSHV的BC3, BCBL1, BCP1有效,butyrate加上反而抑制。Butyrate對EBV的P3HR1(或稱HH514-cl16)及KSHV的BC1有效,TPA有時有 加成作用。另外,NAS上面有一張slide,是PWW以前做BCP1的結果(只有TPA和沒有NaB),請你把它加入你今天報告的slide中,以後就 不用老是在心中掛念著這一件事。
2008/11/18 Lab Meeting 心得彙整
小嬿: 成 功建立293TetgZ三株(B95-8strain),293TetcZ二株 (Akata strain),293TettZ (BC1 strain)四株。將進一步做較細部characterization (leakiness, expression level and growth rate)。Dox請處理48小時以上注意有無growth arrest現象。原則上試圖將maxi-EBV or Akata-EBV送入相對應的Z inducible cell line中。記得與懿宸學長學習(1)穿染Maxi-EBV至細胞中的方法(2)檢測culture supernatant病毒顆粒的方法(PCR of EBV-W)。謝謝。
Team-Wrok部份:經過各位連日來的辛勤耕耘,今天會和小嬿抽取一批小丸子prepare的sample,路過打招呼時請注意不要濺太多RNase給我們。3Q and good luck to us!
2008/12/02 Lab Meeting 心得彙整
SFL去台北參加學生進度報告,所以沒有參加這次meeting。小嬿說 他報告三種Z 在B細胞中再活化EBV之功能(學長問及的問題簡答如下:tZ是由BC1 strain clone出來的,要問是否有negative regulator功能最好是回去同一種細胞裡,將full-length clone出來再做比較比較恰當。不過這部份目前不是我們的重點。會有這個發現是證明了小嬿具有明察秋毫、鍥而不捨的精神,並且也在磨鍊他cDNA cloning的功夫)。
2008/12/10 Lab Meeting 心得彙整
小嬿：(1)EREV8 Dox、T、S、TS、gZ, cZ, tZ準備收尾(利用Akata Z > B958 Z之特性證明外送Z叫不醒endogenous Z;也想知道enodgenous R有被叫出嗎?) (2)準備下週測量EREV8/MaxiEBV viral titer事宜。(3)已pool三株293TetgZ,繼續培養293TetcZ,等有可用的infectious EBV,準備做EZEV。也想嚐試EZKV哩。(4)準備做HONE1TetgZ and TW01TetgZ. (5) Microarray data偷看中…等SFL借到軟體再好好分析。(你要做得完的話,我叫你超人,而且有賞﹍)
開始來問幾個問題,第一有病毒在時,cell也是停在G1嗎?有病毒在時p21,p27,CCNE,CDK4, CDK6, CCND1表現如何呢?在細胞核裡sub-cellular localization有沒有變化呢?
2008/12/16 Lab Meeting 心得彙整
小嬿:著手測試由ntu來的4株293TetER_MaxiEBV是否可產病毒(再提醒一次,做出來一定有賞)。
2008/12/23 Lab Meeting 心得彙整
小嬿: Set up procedures for EBV viral particle detection.
小嬿: Set up procedures for EBV viral particle detection.
2008/12/30 Lab Meeting 心得彙整
小嬿:成功地建立出小量測量supernatant virion DNA的SOP.棒極了.SFL給妳拍拍手(學長們真是難以取悅呀,世界正在改變﹍).往下是來定有多少infectious particle.
2009/01/06 Lab Meeting 心得彙整
小嬿:EBNA1 IFA測試,開始bioinformatics實驗(學Partek package)。一般而言,沒有入門課可上的話就是讀manual。心情放鬆,頭腦保持清楚,學做宅男宅女!
2009/01/20 Lab Meeting 心得彙整
小嬿：以PBMC immortalization及感染293輔以藥篩兩個方法測試dox-EV8/ID4/3A5之infectivity.
陳P:練習用少量的錢、一個RA當酵頭,衝出一點好成績!亞鈴型拉絲雙核
BFB (breakage-fusion-bridge)、terminase、cytokinesis.
2009/02/03 Lab Meeting 心得彙整
小嬿:Survey of Z and EBNA1 expression in 293 cells infected with viral particles prepared from dox-treated EREV8 and MaxiEBVs.
小嬿:Survey of Z and EBNA1 expression in 293 cells infected with viral particles prepared from dox-treated EREV8 and MaxiEBVs.
2009/02/10 Lab Meeting 心得彙整
小嬿:(1)測試EBNA1大寶2血清。(2)設立PCR-based titrating KV virion之系統。(3)準備測試EV/KV lytic replication所處的cell cycle stage.
2009/02/12 by SFL
細胞老化和病毒再活化
這 部份實驗是承接293TetER之後的故事,我希望剛開始的一些結果可以當做小嬿的論文。小蓮請先協助,小丸子與Ingrid在忙完KR後也要漸漸加進 來,希望能夠把這部份的因果關係釐清清楚。以EREV8而言雖然和293TetER所表現flag-ER的量是相似的,但是因有病毒基因套在裡面,所以估 計還不到SA-b-Gal最強的第十天,細胞可能因病毒大量製造的摧殘就掛了。ERKV可能也是如此。所以,我們要弄清楚什麼呢?
⓵Dox-treated 293TetER停在G1,那有病毒的細胞呢?
⓶Dox-treated 293TetER在serum-free這個stress存在下細胞巨觀並未如對照組產生顯著變化,而且病毒再活化時,在48小時以後細胞明顯變圓。再 來,microarray顯示Rta或Rta+EBV或Rta+KSHV均會讓一群控制細胞架構的基因表現異常。另外,小龜的結果又顯示ER一表現可持續 活化Erk(一個和cytoskeleton有密切關連的分子),這些線索之間相互間的關係到底是什麼?
⓷Dox-treated EREV8和ERKV有走完裂解期嗎?製造出來的病毒有感染力嗎?效價好嗎?(小嬿你真的要準備吃大餐了)
⓸另外幾組異常表現的基因如DUB/protease,chromosome binding protein等,如何和老化及病毒再活化連起來呢?
⓹ 細胞老化已知的pathway(growth arrest, activation of mTOR, secretion of mitogenic/antiapoptotic factor, activation of proteosome)可提供病毒什麼好處、讓它引發lytic cycle?
⓶Dox-treated 293TetER在serum-free這個stress存在下細胞巨觀並未如對照組產生顯著變化,而且病毒再活化時,在48小時以後細胞明顯變圓。再 來,microarray顯示Rta或Rta+EBV或Rta+KSHV均會讓一群控制細胞架構的基因表現異常。另外,小龜的結果又顯示ER一表現可持續 活化Erk(一個和cytoskeleton有密切關連的分子),這些線索之間相互間的關係到底是什麼?
⓷Dox-treated EREV8和ERKV有走完裂解期嗎?製造出來的病毒有感染力嗎?效價好嗎?(小嬿你真的要準備吃大餐了)
⓸另外幾組異常表現的基因如DUB/protease,chromosome binding protein等,如何和老化及病毒再活化連起來呢?
⓹ 細胞老化已知的pathway(growth arrest, activation of mTOR, secretion of mitogenic/antiapoptotic factor, activation of proteosome)可提供病毒什麼好處、讓它引發lytic cycle?
2009/02/17 Lab Meeting 心得彙整
小嬿:IFA調件測試、大寶2血清測試。
2009/03/03 Lab Meeting 心得彙整
小嬿:大寶2 EBNA1 staining成功!EREV8 growth rate結果確定。
小嬿:大寶2 EBNA1 staining成功!EREV8 growth rate結果確定。
2009/03/17 Lab Meeting 心得彙整
小嬿: (1) Titration of viral particles released from EREV8: (a) PCR of encapsidatd viral DNA (b) EBNA1 (IFA). (2) Induction rate of EREV8 (a) Flag-R (b) ZEBRA.
2009/03/31 Lab Meeting 心得彙整
小嬿:Induction of Rta, Zta and VCA expression in Dox-treated EV cells.
2009/04/14 Lab Meeting 心得彙整
小嬿:PBCV1 vSET inhibits host transcriptome by global histone methylations.
2009/06/02 Lab Meeting 心得彙整
小嬿:將p2089送入TW01TetER,放大篩選中.
2009/06/23 Lab Meeting 心得彙整
小嬿: Progress report on Rta mediated transactivation of KV promoters.
2009/07/08 Lab Meeting 心得彙整
小嬿: Characterization of TW01TetER-KV clones. None of them can be reactivated by Dox (有二個可能原因(i)能被活化的早在infection時被留在viral sup的少量dox搞砸了 (ii) TW01TetER中,ER表現量不足.
2009/08/04 Lab Meeting 心得彙整
小嬿: Review of 293Tet_Flag_SOX and its EIK mutant. Thanks.
2009/08/18 Lab Meeting 心得彙整
小嬿:〝determination of EREV8 induction rate by IFA and flow cytometry analysis〞出師記。
2009/09/01 Lab Meeting 心得彙整
小嬿:Progress report of TW01Tet/rKSHV.219 : (1) can’t be reactivated by either Dox nor TS, (2) TW01TetER clone 7 has effects in the down-regulation of CDK6 and phosphorylated Rb.
2009/10/13 Lab Meeting 心得彙整
小嬿: Determination of virus titers for EV and KV cells – 不好做的重要實驗,請加油,很接近了呦!
2009/11/03 Lab Meeting 心得彙整
小嬿: Determine the virus titer of ERKV. 以1:4稀釋virus sup的條件下, flow cytometer及肉眼均可用來計算infectious unit. 惟flow cytometer似乎比肉眼高3~4倍. Good.
2009/11/24 Lab Meeting 心得彙整
小嬿: Determine the virus titer of ERKV. 以1:4稀釋virus sup的條件下以肉眼計算.完成三次平均值的實驗. Flow部份暫時擱下, control組background很高.
小嬿: Determine the virus titer of ERKV. 以1:4稀釋virus sup的條件下以肉眼計算.完成三次平均值的實驗. Flow部份暫時擱下, control組background很高.
2009/12/15 Lab Meeting 心得彙整
小嬿: 確 定不發光的EREVp2089、TW01EREVp2089中EBNA1是否還在. Viral genome的stability是目前YJC最care的事,加油! (報告得很清楚喔→你才一講完,大家就紛紛地說出你下一個步驟應該怎樣怎樣、是不是可以用xxx取代xxx云云)
2009/12/29 Lab Meeting 心得彙整
Hammerschmidt的新作在此.
小嬿: 報paper: EBV進入B細胞和上皮細胞後,命運不相同.. 這裡.
謝謝你們! Good Job, 希望自己也有收穫(不懂處,讚嘆處,崇拜處,不認同處..請多利用comment來討論,下年度我們幾乎都會玩到這些東西,所以講者聽者都可以踴躍發言)
2010/01/12 Lab Meeting 心得彙整
小嬿: (1) 報paper: EBV 在鼻咽癌細胞中遺失過程.這裡. (2) 整理EREVp2089, TW01-EREVp2089之進展. Great!! Thank you~
2010/01/26 Lab Meeting 心得彙整
小嬿: PCR detection of EGFP and Hygromycin genes lost in EREVp2089 #B2. EBV induced GI in LCL. 這裡 已email EBV及phil.
2010/02/23 Lab Meeting 心得彙整
小嬿: TW01-EREVp2089 clone #3 的subcloning. 共挑11個pure, green colony, 可惜只測到Z, EA-D起不來, 目前原因仍不明白.
2010/03/09 Lab Meeting 心得彙整
小嬿: (一)補齊paper圖一之western blot analysis, 水喔。 (二)EV titer部份,決定先放棄IFA法測試Z或EBNA之表現,改以q-PCR來訂定medium中virus particle之釋放量。
2010/03/30 Lab Meeting 心得彙整
小嬿: 整理報告建立TW01-EREVp2089_Fast and Slow之過程. 好棒! 妳的細胞株一定會有許多人想要的 (要不要來patent? 哈哈,想paper比較要緊)
小嬿: 整理報告建立TW01-EREVp2089_Fast and Slow之過程. 好棒! 妳的細胞株一定會有許多人想要的 (要不要來patent? 哈哈,想paper比較要緊)
2010/04/13 Lab Meeting 心得彙整
小嬿: (一)補齊paper圖一之virus particle kinetics。 (二)確定TWO1-EREVp2089有infectious particle 產出。2010/05/04 Lab Meeting 心得彙整
小嬿: 報paper:LSD1對HSV1/VZV immediate-early gene promoter上H3K4/H3K9之影響。Thank you~
2010/05/18 Lab Meeting 心得彙整
小嬿: 報paper critiques.謝謝大家的鼓勵.We can make it!
2010/05/26 Lab Meeting 心得彙整 (未來目標)
小嬿:(1) 293/rKSHV.219與BC1中KSHV latent genome被Rta活化情形. (2) EREV8 及 ERKV viral particles之titration,以及其中Dox 96h infectious unit之訂定. (3) 以Na-butyrate及Dox處理EREV8細胞後,Rta與Zta之表現程度比較.(4)expression kinetics of cell cycle regulators between Dox 3h-Dox 72h in 293TetER, EREV8 and ERKV cells. (5) Latent genome copy number in EREV8, ERKV and TW01EREVp2089_S and _F.
2010/06/08 Lab Meeting 心得彙整
小嬿: Expression kinetics of cell cycle regulators in TW01-EREVp2089_F and _S.
2010/06/22 Lab Meeting 心得彙整
小嬿: 彙整Rta-mediated cell cycle arrest vs EV/KV reactivation kinetics。 Good and Thanks。
2010/07/20 LabMeeting 心得彙整
小嬿: 彙 整(1) Lytic replication “throughput” in TPA/Butyrate- versus Dox-treated EREV8 cells. (2) Raji EA-D inducing unit assay. (3) Dox 96h-1A4: viral particle (~0.78 M / ml), 其中約6%可感染Raji而3%可感染293。 Good and Thanks。
2010/08/04 LabMeeting 心得彙整(自己Lab開)
小嬿:討論cell cycle analysis Flow的詳細步驟。
2010/09/01
介紹在英國看到的兩篇poster (from Dr. Tsao)
1. HONE1-EBV可不受外物刺激就自動產生病毒, HK1-EBV 需要外物刺激, NP460hTert-EBV推測是abortive reactivation(未知停在哪). 在TNF-alpha刺激的HK1-EBV, BMRF1的表現量與Qp相反, 似乎與BZLF1的量不成正相關.
2. 有EBV存在的細胞株在營養缺乏的環境下較有利生存
1. HONE1-EBV可不受外物刺激就自動產生病毒, HK1-EBV 需要外物刺激, NP460hTert-EBV推測是abortive reactivation(未知停在哪). 在TNF-alpha刺激的HK1-EBV, BMRF1的表現量與Qp相反, 似乎與BZLF1的量不成正相關.
2. 有EBV存在的細胞株在營養缺乏的環境下較有利生存
2010/10/27
1. Establishment of several stable cell lines: Akata-p2089, TW01EREV-Akata/EGFP, TW03Tet, and TW05Tet. I need YCK’s help to screen TW03Tet and TW05 Tet cells. I’ll transfer these clones to YCK tomorrow.
2. To test sonication condition of ChIP.
2010/11/17
1. Establishment of Akata-p2089, TW01EREV-Akata-EGFP cell lines.
2. To test the sonication condition of ChIP.
3. To evaluate the expression level of X protein in 293- and TW01-series cell lines.
2010/12/15
1. TW01EREV_S無法用SAHA induction成功–>放棄S
2. 所建立的Akata-p2089, 用hIgG沒有辦法induction(by Q-PCR), 在induce 96hrs情況下, 看不到Rta, Zta及EAD表現, 但有少量的gp350/220. 根據之前研究, 可續偵測LMP2A的存在與否.
3. Akata-EGFP/EBV在hIgG induce 48hrs可到達plateau (in sup, by Q-PCR).
4. 目前所建立的35個TW01EREV中, EBV Rta表達量高者似乎無法induction, 僅有TW01EREV-26可用NaB產出較高量的病毒; 在EBV Rta表達中量者中, 挑到1個Dox與 NaB效果相當-TW01EREV-27. 但其induction rate, viral particles等細項仍須confirm.
5. 搜尋其他研究者的ChIP sonication 條件.
2. 所建立的Akata-p2089, 用hIgG沒有辦法induction(by Q-PCR), 在induce 96hrs情況下, 看不到Rta, Zta及EAD表現, 但有少量的gp350/220. 根據之前研究, 可續偵測LMP2A的存在與否.
3. Akata-EGFP/EBV在hIgG induce 48hrs可到達plateau (in sup, by Q-PCR).
4. 目前所建立的35個TW01EREV中, EBV Rta表達量高者似乎無法induction, 僅有TW01EREV-26可用NaB產出較高量的病毒; 在EBV Rta表達中量者中, 挑到1個Dox與 NaB效果相當-TW01EREV-27. 但其induction rate, viral particles等細項仍須confirm.
5. 搜尋其他研究者的ChIP sonication 條件.
2010/12/07 Lab Meeting 心得彙整
小嬿: 報告Vieira新作：(1) keratinocyte (角質細胞) vs. epithelial cells (上皮細胞) (2) keratinized vs. non-keratinized (3) simple epithelium vs. stratified epithelium. 哇，SFL覺得這部份實在有必要再回去讀教科書一下，越讀越複雜？！
2011/01/12
1. 偵測Dox-treated TW01-EREV-26/27的
a. viral particles, 似乎隨時間增加, 但infectivity待測
b. viral protein expression pattern比EREV8稍快
c. 27的viral particles production比26高
d. 此次的NaB效果遠比Dox低, 可多試NA或EREV8當positive control, 確定NaB效果
e. 細胞數少的induction efficiency較好
a. viral particles, 似乎隨時間增加, 但infectivity待測
b. viral protein expression pattern比EREV8稍快
c. 27的viral particles production比26高
d. 此次的NaB效果遠比Dox低, 可多試NA或EREV8當positive control, 確定NaB效果
e. 細胞數少的induction efficiency較好
2. ChIP的sonication條件:
fixation mode: suspension
cell conc: 1.5 x 10^7 cells/ml,
sonication vol: 150 ul/reaction
sonication cycle: 30″ ON, 30″ OFF, for 10 cycles
fixation mode: suspension
cell conc: 1.5 x 10^7 cells/ml,
sonication vol: 150 ul/reaction
sonication cycle: 30″ ON, 30″ OFF, for 10 cycles
3. RTS-15與Flag-Rta送入293細胞後的表達量相當, 但加MG115之後未stablize Rta的量
2011/01/26
1. The stability of EBV Rta
a. the expression level of RTS-15 and Flag-Rta is similar but slightly higher than Rta-V5.
b. Rta expression level of 6-hr transfection is blocked by MG115 and fragmented into smaller size, and the fragments are stablized by MG115 treatment.
2. The expression level of Akata-EGFP/EBV and Akata-p2089 is similar.
3. Higher titer of viral particles were detected in long-term induction of Akata-EGFP/EBV but the infectivities were decreased.
4. Profiling of TW01-EREV-27
-Induction rate is correlated to cell density. Lower is better.
-to be continued.
a. the expression level of RTS-15 and Flag-Rta is similar but slightly higher than Rta-V5.
b. Rta expression level of 6-hr transfection is blocked by MG115 and fragmented into smaller size, and the fragments are stablized by MG115 treatment.
2. The expression level of Akata-EGFP/EBV and Akata-p2089 is similar.
3. Higher titer of viral particles were detected in long-term induction of Akata-EGFP/EBV but the infectivities were decreased.
4. Profiling of TW01-EREV-27
-Induction rate is correlated to cell density. Lower is better.
-to be continued.
2011/03/02
1. check Q-PCR primers: p21 and SFN–>Workable!
2. ChIP test :
a. antibodies: mouse IgG, CTCF and Flag antibody
b. primers: H19ICR, SFN, GAPDH(coding region)
c. results: (1) the problem of sonication is not dissolved
(2) condition of Q-PCR is not well, but standard end-point PCR is OK
(3) input is not equal. (one of them is disappeared, actually)
(4) the amount of DNA fragment bound by mouse IgG was similar to CTCF
d. Future work:
(1) to try magnetic system by using standard end-point PCR (IgG and CTCF)
(2) to test Q-PCR condition
2. ChIP test :
a. antibodies: mouse IgG, CTCF and Flag antibody
b. primers: H19ICR, SFN, GAPDH(coding region)
c. results: (1) the problem of sonication is not dissolved
(2) condition of Q-PCR is not well, but standard end-point PCR is OK
(3) input is not equal. (one of them is disappeared, actually)
(4) the amount of DNA fragment bound by mouse IgG was similar to CTCF
d. Future work:
(1) to try magnetic system by using standard end-point PCR (IgG and CTCF)
(2) to test Q-PCR condition
2011/03/16
To test Magnetic ChIP method:
1. non-specific band was less than agarose method
2. By using standard end-point PCR analysis, RNA Pol II is able to bind promoters of GAPDH, H19, and SFN; CTCF can bind H19 promoter and weakly on SFN promoter.
3. The problem of sonication efficiency is not dissolved yet.
1. non-specific band was less than agarose method
2. By using standard end-point PCR analysis, RNA Pol II is able to bind promoters of GAPDH, H19, and SFN; CTCF can bind H19 promoter and weakly on SFN promoter.
3. The problem of sonication efficiency is not dissolved yet.
2011/04/06
1. 降低細胞數至7.5 x 10^6/ml做sonication的效果仍未達百分百, 將試5 x 10^6/ml的效果
2. 比較TW01TetER(19) Ctrl-24hrs及Dox-24hrs, (1) CTCF bind在H19及SFN promoter的能力似乎相當, 一般PCR及Q-PCR所看到的差距不大 (2) 初步測試Rta也可bind在H19(weak)及SFN promoter上, Q-PCR 確認中
2011/05/03
1. 比較Dox- 和TPA/NaB-treated TW01-EREV-26(ER-high)/2716(ER-medium):
a. the amount of spontaneous lytic and the responses to inducers: 2716 > 26
b. Dox在2716的反應比TS佳, 但在26的反應比TS差
c. 未比較protein expression level
2. 比較Dox-treated TW01-EREV-26/2716(0-6天):
a. viral proteins: Flag-Rta/total Rta與之前結果相同: 26>2716; BZLF1, BMRF, BLLF1在兩細胞株都以類似EREV8的cascade pattern出現, 但表現量2716>26; latent protein, EBNA1在Dox處理後也有稍稍增多的現象, 2716較為明顯
b. viral particles: 24小時未有病毒量的變化, 48小時開始induce出病毒, 26細胞株所產病毒量約在5-10x 10^4/ml, 而2716的72小時約為10^6/ml, 72-144小時的病毒量相差不大
c. cell death associated markers: 目前試了caspase-3及PARP-1. caspase-3的cleavage form在26較多, 但在induce 3天之後便減少(in both cells); PARP-1的pattern類似於caspase-3.
d. cell cycle associated proteins: (G1) CDK4/6, CCND1, CCND3下降; p21, p27, p53, SFN上升; CCNE1變化不明顯; (S) CCNA, E2F1, p-pRb, pRb下降
a. the amount of spontaneous lytic and the responses to inducers: 2716 > 26
b. Dox在2716的反應比TS佳, 但在26的反應比TS差
c. 未比較protein expression level
2. 比較Dox-treated TW01-EREV-26/2716(0-6天):
a. viral proteins: Flag-Rta/total Rta與之前結果相同: 26>2716; BZLF1, BMRF, BLLF1在兩細胞株都以類似EREV8的cascade pattern出現, 但表現量2716>26; latent protein, EBNA1在Dox處理後也有稍稍增多的現象, 2716較為明顯
b. viral particles: 24小時未有病毒量的變化, 48小時開始induce出病毒, 26細胞株所產病毒量約在5-10x 10^4/ml, 而2716的72小時約為10^6/ml, 72-144小時的病毒量相差不大
c. cell death associated markers: 目前試了caspase-3及PARP-1. caspase-3的cleavage form在26較多, 但在induce 3天之後便減少(in both cells); PARP-1的pattern類似於caspase-3.
d. cell cycle associated proteins: (G1) CDK4/6, CCND1, CCND3下降; p21, p27, p53, SFN上升; CCNE1變化不明顯; (S) CCNA, E2F1, p-pRb, pRb下降
Future work:
1. 釐清cell death與viral reactivation之間的順序 (by TUNEL assay?)
2. 除了apoptosis之外, 可觀察其他如autophagy相關markers
1. 釐清cell death與viral reactivation之間的順序 (by TUNEL assay?)
2. 除了apoptosis之外, 可觀察其他如autophagy相關markers
2011/06/01
1. 設計12個target gene的CTCF binding site primers. 初步用ERKV及Raji的genomic DNA測試primer, 九組較佳, 三組較弱, 一組沒有p出來. –>後面這四組primer會再做進一步確認condition
2. 利用IP觀察CTCF是否跟Rta有interaction. IP CTCF後有抓到Rta, 但其抓到的量與Dox induction與否沒有關係(偽Rta?). (使用ICON的blocking reagent後, IgG的band有比較乾淨.)
3. 利用Thermo的fraction kit區分TW01TetER-19細胞的cytosol及nuclear protein. PARP1及CTCF乾淨地位於核內; Rta, p53, H3在cytosol有殘留; GAPDH及a-tubulin主要在質; 怪的是PCNA大部分在細胞質.
2. 利用IP觀察CTCF是否跟Rta有interaction. IP CTCF後有抓到Rta, 但其抓到的量與Dox induction與否沒有關係(偽Rta?). (使用ICON的blocking reagent後, IgG的band有比較乾淨.)
3. 利用Thermo的fraction kit區分TW01TetER-19細胞的cytosol及nuclear protein. PARP1及CTCF乾淨地位於核內; Rta, p53, H3在cytosol有殘留; GAPDH及a-tubulin主要在質; 怪的是PCNA大部分在細胞質.
–>1. 這篇paper指 出位於質內的PCNA具有anti-apoptosis的效用(in neutrophil), 或許這是TW01細胞可以serum starvation還如此快樂的原因之一. (而一點點的PCNA在核內就夠DNA replication 用?或大部分細胞在G1 phase, PCNA不用進核工作)
–>2. 試試沛文的fractionation方法
–>3. 試試IP Flag, 看CTCF是否會被抓下來
–>2. 試試沛文的fractionation方法
–>3. 試試IP Flag, 看CTCF是否會被抓下來
2011/06/29
1. 修正sonication條件為30″ ON, 30″ OFF, 8 cycles, 利用此條件在TW01TetER_16細胞做ChIP. 初步測試myc primer, PCR條件未達到最好: 1) 跑膠結果有很多用剩的primer或dimer; 2) input量很多, 而IP的量很少, 沒有調整好比例; 3) myc-2和myc-3的product跑2% agarose不漂亮
–>end-point PCR 固定template量, 調整input的跑膠量; myc-2 myc-3跑3% agarose gel
–>試試Q-PCR條件
–>end-point PCR 固定template量, 調整input的跑膠量; myc-2 myc-3跑3% agarose gel
–>試試Q-PCR條件
2. 利用Thermo fractionation kit分出TW01TetER_16細胞的nuclear extract做IP, WB結果看到CTCF有抓到疑似Rta的band, 但用Flag抗體IP則看不到CTCF.
–> 將Flag-Luc, ER, KR, gZ送入HEK293細胞中表現24 hrs後收lysate做IP, WB結果看到CTCF可抓到明顯的ER(by 467)和微弱的gZ(by 4F10), 抓不到Luc(by Flag)和KR(by Yoshi’s Ab); 但用Flag IP, 則在四組細胞中都沒有看到CTCF.
–> 將Flag-Luc, ER, KR, gZ送入HEK293細胞中表現24 hrs後收lysate做IP, WB結果看到CTCF可抓到明顯的ER(by 467)和微弱的gZ(by 4F10), 抓不到Luc(by Flag)和KR(by Yoshi’s Ab); 但用Flag IP, 則在四組細胞中都沒有看到CTCF.
2011/08/03
1. 在HEK293分別做Flag-Luc/ER/KR/gZ的transient transfection 24hrs,
a. IP CTCF: 1/6 of IP中, WB CTCF未有band–>用5/6的memebrane check again;
5/6 of IP中看到ER (by 467), 微弱的gZ(by 4F10), 未見KR(by Yoshi’s);
b. IP Flag: 失敗! 1/20 of IP可見Flag蛋白被IP下來, 但未見CTCF!
@WHT建議二抗濃度可再做調整, 讓background更乾淨(e.g. ~100 kD的band)
@在TW01系統試試看!
@SFL建議在293FT做transfection效果更佳! (plus co-co-co-transfection!)
2. 利用ChIP分析TW01TetER_16細胞中, 在Dox induction的狀況下, CTCF所binding的DNA fragment量是否有所改變
–>依據Q-PCR結果顯示, input DNA量不穩定, 影響CTCF IP的數值計算; mock(IgG) IP所得DNA量較少, Ct值SD非常大
@SFL建議做少一點sample量, IP CTCF和Rta, 並且screen先前擬定偵測的gene
a. IP CTCF: 1/6 of IP中, WB CTCF未有band–>用5/6的memebrane check again;
5/6 of IP中看到ER (by 467), 微弱的gZ(by 4F10), 未見KR(by Yoshi’s);
b. IP Flag: 失敗! 1/20 of IP可見Flag蛋白被IP下來, 但未見CTCF!
@WHT建議二抗濃度可再做調整, 讓background更乾淨(e.g. ~100 kD的band)
@在TW01系統試試看!
@SFL建議在293FT做transfection效果更佳! (plus co-co-co-transfection!)
2. 利用ChIP分析TW01TetER_16細胞中, 在Dox induction的狀況下, CTCF所binding的DNA fragment量是否有所改變
–>依據Q-PCR結果顯示, input DNA量不穩定, 影響CTCF IP的數值計算; mock(IgG) IP所得DNA量較少, Ct值SD非常大
@SFL建議做少一點sample量, IP CTCF和Rta, 並且screen先前擬定偵測的gene
GOGOGO! 往PLoS Pathogen邁進!!!
2011/08/31
1. to observe whether CTCF interact with EBV Rta by Immunoprecipitation assay
a) in TW01 inducible cell lines, CTCF and Flag-Rta can not be immunoprecipitated by each other. I also tried Flag beads, which are provided by Sigma, both of Flag-Luc and Flag-ER caught CTCF. I can not prove the interaction between CTCF and EBV Rta in TW01 inducible cell lines.
b) in co-transfected HEK293/293FT cell lines(Flag-Luc/ER/KR), CTCF can immunoprecipitate Flag-ER. However, the ratio of each plasmid is unfair, i will adjust the amount of these plasmids (may ignore Luc) and perform this experiment again.
a) in TW01 inducible cell lines, CTCF and Flag-Rta can not be immunoprecipitated by each other. I also tried Flag beads, which are provided by Sigma, both of Flag-Luc and Flag-ER caught CTCF. I can not prove the interaction between CTCF and EBV Rta in TW01 inducible cell lines.
b) in co-transfected HEK293/293FT cell lines(Flag-Luc/ER/KR), CTCF can immunoprecipitate Flag-ER. However, the ratio of each plasmid is unfair, i will adjust the amount of these plasmids (may ignore Luc) and perform this experiment again.
2. to screen 12 putative target genes in CTCF/Flag-ChIP samples
利用Q-PCR的方式, 這12個genes無法在CTCF-ChIP的samples中被偵測到; 但利用一般PCR的方式, 則可偵測到JUN及H19(雖然band很微弱)
a) 在ChIP之前的sonication片段可能太短, 以至於所設計的這些將近500 bp PCR product的primer不能bind上DNA
b) ChIP的效率不好, 在抗體/磁珠/sample量的比例上該做些調整
利用Q-PCR的方式, 這12個genes無法在CTCF-ChIP的samples中被偵測到; 但利用一般PCR的方式, 則可偵測到JUN及H19(雖然band很微弱)
a) 在ChIP之前的sonication片段可能太短, 以至於所設計的這些將近500 bp PCR product的primer不能bind上DNA
b) ChIP的效率不好, 在抗體/磁珠/sample量的比例上該做些調整
2011/09/28
1. 利用Chromatin IP的sample做IP-WB測試CTCF/Flag/Rta抗體使用量:
(1) CTCF使用cell signaling的rabbit Ab(12 ul)效果較佳
(2) Flag使用cell signaling的rabbit Ab(12 ul)效果較佳, 但抗體未完全bind在磁珠上, 可再降低抗體量
(3) Rta抗體(Argene)黏附於磁珠的效果不佳, 所抓到的Rta量也不多
–> 確定sonication條件之後就可以上述兩個較好的抗體做ChIP!
(1) CTCF使用cell signaling的rabbit Ab(12 ul)效果較佳
(2) Flag使用cell signaling的rabbit Ab(12 ul)效果較佳, 但抗體未完全bind在磁珠上, 可再降低抗體量
(3) Rta抗體(Argene)黏附於磁珠的效果不佳, 所抓到的Rta量也不多
–> 確定sonication條件之後就可以上述兩個較好的抗體做ChIP!
2. 測試Akata-EGFP/EBV產出的病毒infect HEK293細胞的效率
(1) 以IgG-48hrs的效果最好, 但所看到GFP(+)的細胞大部分都不健康–>調整病毒濃縮的倍數, 看看是不是病毒太多, 或PEG太多讓細胞有毒性
(2) 將infect 48hrs的細胞收下測得微弱的Rta跟size較小的Zta–> Z若未經modification應為29kDa, 用western看到Akata strain的Z約38kDa, 比B95.8大(約36kDa); 而truncated form最小, 約31kDa(推算); 之後可收RNA, 看所測到的Z是否有truncation.
(1) 以IgG-48hrs的效果最好, 但所看到GFP(+)的細胞大部分都不健康–>調整病毒濃縮的倍數, 看看是不是病毒太多, 或PEG太多讓細胞有毒性
(2) 將infect 48hrs的細胞收下測得微弱的Rta跟size較小的Zta–> Z若未經modification應為29kDa, 用western看到Akata strain的Z約38kDa, 比B95.8大(約36kDa); 而truncated form最小, 約31kDa(推算); 之後可收RNA, 看所測到的Z是否有truncation.
2011/11/23
1. 確定ChIP的前置步驟條件:
Cell: 4 x 10^7 cells/ml lysis buffer
Sonication: 45″ ON, 45″ OFF for 10-12 cycles (depend on cell type)
2. 在TW01-EREV_2716細胞中, 觀察到CTCF在Dox處理24小時後結合在cellular promoters上的量比6hrs與未處理Dox的細胞來得多. 顯示 Rta的出現可能讓CTCF對這些promoter的affinity更好. 但是在EREV8細胞中, 同樣的時間點沒辦法看到類似的情況.
Cell: 4 x 10^7 cells/ml lysis buffer
Sonication: 45″ ON, 45″ OFF for 10-12 cycles (depend on cell type)
2. 在TW01-EREV_2716細胞中, 觀察到CTCF在Dox處理24小時後結合在cellular promoters上的量比6hrs與未處理Dox的細胞來得多. 顯示 Rta的出現可能讓CTCF對這些promoter的affinity更好. 但是在EREV8細胞中, 同樣的時間點沒辦法看到類似的情況.
–> 補齊其他cellular promoter的PCR detection.
–> 目前推測TW01EREV-2716走lytic cycle的速度比EREV8來得快, 可能24hr還看不到CTCF在EREV8細胞的變化. 試試在EREV8細胞中觀察48小時的CTCF ChIP.
–> 目前推測TW01EREV-2716走lytic cycle的速度比EREV8來得快, 可能24hr還看不到CTCF在EREV8細胞的變化. 試試在EREV8細胞中觀察48小時的CTCF ChIP.
2012/01/11
1. 為避免viral genome的干擾, 將系統換回TW01TetLuc及TW01TetER_16. 比較Dox處理6, 24, 48小時, CTCF binding在H19 ICR及c-Myc P2 promoter位置的量. Q-PCR結果顯示在兩個細胞中24小時CTCF結合在H19 ICR量比6小時多,並且在48小時下降; 在P2 promoter的位置, Luc細胞在三個時間點的結合量相近, ER細胞在24小時結合量上升, 48小時下降. 但, 兩組CTCF結合位置在Luc組細胞都比ER組來得多
–>加做Ctrl-6hr觀察Luc/ER兩組細胞的background差異
2. 整理過去paper報導CTCF在EBV/KSHV genome上的binding sites(CBS)
–> EBV genome上約有20個位點, KSHV約有10個位點, 兩者的胃點有些有相似
–> 先focus在LCR, K-RTA/BZLF1 promoter, 及OriLyt等位置, 準備設計primers
2012/05/09
1. 欲確定在293TetLuc細胞中, Dox treatment 3-9hrs之間, CTCF結合在c-Myc/H19 promoter的pattern. 初步CTCF-ChIP assay結果顯示在兩個promoter上, CTCF的結合量未有明顯的改變. 之後將進行293TetER細胞的CTCF-ChIP assay.
2. 分析目前已知Rta會結合的promoters, 利用軟體預測Rta responsive element(RRE)/CTCF binding site(CBS)/sp1 binding site. 目前觀察到的情形如下:
a) CTCF 與Rta結合位置重疊: BALF2, SFN
b) CTCF 與Sp1結合位置重疊: p21, SFN
c) CTCF/Rta/Sp1結合位置是鄰居: BLLF3, BHRF1/BHLF1
分析結果未整理完全, 待續
a) CTCF 與Rta結合位置重疊: BALF2, SFN
b) CTCF 與Sp1結合位置重疊: p21, SFN
c) CTCF/Rta/Sp1結合位置是鄰居: BLLF3, BHRF1/BHLF1
分析結果未整理完全, 待續
3. 利用direct infection的方式建立攜帶螢光的293LucEV/293EREV. 所篩到的clone能被Dox induction出來的viral particles比EREV8少了至少10倍. 之後提高G418濃度也未能讓viral particles被release更多, 因此先停住這個實驗.
2012/09/19
1. 欲觀察CTCF是否在EBV/KSHV的潛伏期中扮演repressor的角色:
a) 利用shRNA-lentivirus infection方式knockdown CTCF expression五天之後, 發現病毒RNA/protein表現量上升, 存在於細胞中及release到上清液的的病毒DNA copies也變多.
–> shCTCF clone B情況較特殊, 將mix其他三個clone的病毒液再觀察一次結果
–> viral transcripts/protein可多看BMRF1, K-bZIP
b) 將CTCF表現質體送入細胞中表現3天, 發現病毒RNA/protein表現量沒有變化, 存在於細胞中及release到上清液的病毒DNA copies變化也不大
–> 調整細胞濃度做DNA transfection再觀察一次
a) 利用shRNA-lentivirus infection方式knockdown CTCF expression五天之後, 發現病毒RNA/protein表現量上升, 存在於細胞中及release到上清液的的病毒DNA copies也變多.
–> shCTCF clone B情況較特殊, 將mix其他三個clone的病毒液再觀察一次結果
–> viral transcripts/protein可多看BMRF1, K-bZIP
b) 將CTCF表現質體送入細胞中表現3天, 發現病毒RNA/protein表現量沒有變化, 存在於細胞中及release到上清液的病毒DNA copies變化也不大
–> 調整細胞濃度做DNA transfection再觀察一次
2. 利 用ChIP方式觀察EBV Rta表現是否影響CTCF結合於病毒基因體的能力. 初步結果可觀察到Dox-48hr細胞中, CTCF binding量比non-induction少; 而Rta除了binding在已知的RRE之外, 在目前所偵測的DNA片段上也都有Rta的訊號.
–> EREV8的ChIP訊號都比ERKV弱, 需要多加確認
–> ChIP所得DNA量太少, 將做些調整以提高所得DNA濃度
–> EREV8的ChIP訊號都比ERKV弱, 需要多加確認
–> ChIP所得DNA量太少, 將做些調整以提高所得DNA濃度
2014/03/12
Fig. 1. CTCF repressed the lytic cycle replications of EBV and KSHV in viral latently infected cells.
> (a). The band of CTCF ectopic-expressed cells is over-exposed.
> (b). The expression level of shCTCF is similar to shLuc.
> (a). The band of CTCF ectopic-expressed cells is over-exposed.
> (b). The expression level of shCTCF is similar to shLuc.
Fig. 2. Rta binding sites are similar to CTCF binding sites
Fig. 3. 4. EBV Rta expression increased DNA methylation on the target cellular promoter thus interfered with the occupancies of CTCF.
> (a). RNA expression kinetics of cellular genes should be showed in parallel.
> (b). To confirm the methylation states of cellular promoters in EREV8 cells, especially at 24 h of Dox induction.
> (a). RNA expression kinetics of cellular genes should be showed in parallel.
> (b). To confirm the methylation states of cellular promoters in EREV8 cells, especially at 24 h of Dox induction.
Fig. 5. Rta expression induced CpG methylation on LCR, RCR, and oriLyt region in both EBV and KSHV genomes that influenced CTCF bindings.
> (a). RNA expression kinetics of viral genes around LCR/RCR/oriLyt region should be showed in parallel.
> (b). To move the model figure to the last one.
> (c). To confirm the CTCF binding on EBV RCR.
> (d). To confirm the DNA methylation states of EBV LCR, KSHV RCR
> (a). RNA expression kinetics of viral genes around LCR/RCR/oriLyt region should be showed in parallel.
> (b). To move the model figure to the last one.
> (c). To confirm the CTCF binding on EBV RCR.
> (d). To confirm the DNA methylation states of EBV LCR, KSHV RCR
Fig. 6. EBV Rta associated with DNA methyltransferase, DNMT3A and DNMT3B.
> In Fig. 6B and 6C, the expression level of viral immediate-early proteins were not equal. In addition, normal rabbit IgG should not interact with any viral proteins. It is necessary to improve the wash step through IP experiment.
> In Fig. 6B and 6C, the expression level of viral immediate-early proteins were not equal. In addition, normal rabbit IgG should not interact with any viral proteins. It is necessary to improve the wash step through IP experiment.
| Date | Title |
| 05/06/12 | 小嬿: ChIP assays of CTCF on c-Myc/H19 promoters in Dox-treated 293TetLuc and 293TetER cells. |
| 05/06/12 | 小嬿: Luciferase protein did not affect the binding of CTCF on both promoters. |
| 05/06/12 | 小嬿: EBV Rta slightly decreased the binding of CTCF on c-Myc promoter, but had no effect on H19. |
| 05/23/12 | 小嬿: To test the ChIP condition for Flag antibodies。 |
| 05/23/12 | 小嬿: The results indicated that the Flag antibody from Turbid-River might immunoprecipitate non-specific DNA fragments。 |
| 05/30/12 | 小嬿: Result of IP efficiency: GE Mag Sepharose is much better than Millipore Magnabeads. The next step is to test the ChIP ability。 |
| 05/30/12 | 小嬿: The talk “Identifying biologically interpretable transcription factor knockout targets by jointly analyzing the transcription factor knockout microarray and the ChIP-chip data” by Prof. Wei-Sheng Wu was interesting! |
| 06/06/12 | 小嬿: Flag-ChIP by using GE Mag Sepharose did not work, the second round is on going。 |
| 06/27/12 | 小嬿: Although the knockdown efficiency of shCTCF is not optimal, |
| 06/27/12 | 小嬿: CTCF knockdown seemed to enhance EBV lytic reactivation in Dox-treated EREV8 cells. |
| 07/04/12 | 小嬿: Try to find a better way in enhancing shCTCF efficiency . |
| 07/11/12 | 小嬿: To arrange data for poster presentation. |
| 08/01/12 | 小嬿: 參加在U. Penn舉行的第一屆ICOHAD, 充電中… |
| 08/22/12 | 小嬿：生產TW01-ERGV+Dox 96hrs病毒液 |
| 08/22/12 | 小嬿：利用ChIP方式來確定CTCF/Rta binding於KV的趨勢 |
| 08/01/12 | 小嬿：以ERKV+Dox48hrs觀察CTCF及Flag-Rta結合於DNA上的量. |
| 08/01/12 | 小嬿：改變CTCF表現量以觀察CTCF對於virus進入lytic cycle的影響 |
| 09/05/12 | 小嬿：LTK所製造的Rta抗體可拿來做IP. |
| 09/26/12 | 小嬿：利用軟體分析BARF1與BALF2兩基因之間的序列, 發現其中有兩段序列含有Rta與CTCF的結合位點, 可設計primer分析之 |
| 10/03/12 | 小嬿：Construction of pCMV6-AC-deltaCTCF |
| 10/31/12 | 小嬿：表現CTCF於EREV8與ERKV細胞似乎有壓制病毒進行reactivation的進行(protein level和病毒DNA複製量) |
| 10/31/12 | 小嬿：利用shRNA lentivirus方式抑制CTCF在EREV8及ERKV細胞中表現量, 有較多的病毒進入reactivation(RNA/protein level和病毒DNA複製量) |
| 11/07/12 | 小嬿：To observe the expression level of CTCF in Dox-treated cells |
| 11/14/12 | 小嬿：Protein expression level of CTCF was slightly decreased in Dox-treated EREV8 cells for 48 hrs |
| 11/21/12 | 小嬿：To observe the myc and H19 transcription levels in CTCF-depleted 293KV cells. |
| 11/28/12 | 小嬿：分析CTCF所調控的基因promoter上是否有Rta binding site |
| 12/11/12 | 小嬿：整理CTCF調控基因表現的機制(進行中, 完成後再與大家分享), 並且設計Rta可能參與調控區域的primers |
| 12/18/12 | 小嬿：針對CTCF所調控基因設計Rta可能干擾區域的primer. 初步測試結果除CCND1之外, 其他primer可用. 將再對CCND1的部分改進. |
| 01/24/13 | 小嬿：針對已知CTCF調控基因的位點做ChIP assay, 看到在Dox處理(+PAA)48小時的EREV8細胞中CTCF結合於DNA的量皆下降的情況. 正著手重複此實驗以確定此下降趨勢, 同時觀察Rta蛋白結合 |
| 01/30/13 | 小嬿：Confirme the decreased pattern of CTCF binding on the human/viral genomes. |
| 01/30/13 | 小嬿：To test the binding ability of EBV Rta on the human/viral genomes. |
| 04/03/13 | 小嬿：Ｄesign primers for point mutations on EBV Rta DNA binding domain. (K213A & K156A) |
| 04/17/13 | 小嬿：To observe the DNA binding pattern of EBV Rta and CTCF in Dox-treated and untreated EREV8/ERKV cells (12/24h). |
| 04/17/13 | 小嬿：To design the primers for ChIP assays on the latency/lytic control regions on EBV/KSHV genomes. |
| 04/24/13 | 小嬿：To observe the EBV Rta/CTCF binding patterns on the viral genomes in Dox-treated EREV8/ERKV cells. |
| 04/24/13 | 小嬿：Rta bound the LCR and the upstream of the immediated-early genes on both of EBV and KSHV genomes in 12h-Dox treated cells, however, |
| 04/24/13 | 小嬿： CTCF dropped from these regions until 24-48 h after Dox induction. |
| 05/08/13 | 小嬿：To observe the CTCF binding kinetics in Dox-treated EREV8/ERKV cells. |
| 05/08/13 | 小嬿：The occupancies of CTCF on the cellular/viral genomes were decreased in a time-dependent manner. |
| 05/15/13 | 小嬿：Cellular gene expressions in CTCF over-expression/knockdown/Rta-induced EVKV cells. |
| 05/22/13 | 小嬿：Rta-mediated cellular genes expression kinetics in Dox-treated EREV8 and ERKV cells. |
| 05/29/13 | 小嬿：Rta-mediated cellular genes expression kinetics in Dox-treated EREV8 and ERKV cells. |
| 06/05/13 | 小嬿：Rta-mediated cellular genes expression kinetics in Dox-treated EREV8 and ERKV cells. |
| 06/05/13 | 小嬿：CTCF and EBV Rta binding kinetics on the EBV genome in Dox-treated EREV8 cells. |
| 06/19/13 | 小嬿：CTCF and EBV Rta binding kinetics on the EBV genome in Dox-treated EREV8 cells。 |
| 07/03/13 | 小嬿：The preliminary results indicated that the DNA binding mutant of EBV Rta, K156A, failed to regulated genes expressions. |
| 07/10/13 | 小嬿：Rta DNA binding mutant K156A failed to regulate MYC, CDKN1A, SFN, and BZLF1/K-RTA. |
| 07/30/13 | 小嬿：Rta K156A mutatnt failed to regulate CCND1/SFN and viral immediate-early proteins in EREV8 and ERKV cells. |
| 07/30/13 | 小嬿：Rta increased the luciferase activity of c-Myc promoter. |
| 08/07/13 | 小嬿：In Dox-treated ERKV cells, increased methylations were observed on the CCND1 promoter, c-Myc P2 promoter and c- Myc boundary region compared to the untreated cells. |
| 08/14/13 | 小嬿：Latency control region (LCR) and reactivation control region (RCR) of KSHV genome was methylated after EBV Rta expression for 48 h. |
| 09/04/13 | 小嬿：整理Rta表現之後, EREV8和ERKV細胞中的細胞及病毒基因體甲基化情形。 |
| 10/09/13 | 小嬿：To exam whether EBV Rta interacts with DNMTs by co-IP analysis. |
| 10/09/13 | 小嬿：In preliminary results, EBV Rta interacts with DNMT3a/3b whereas KSHV K-RTA interacts with DNMT3b. |
| 10/30/13 | 小嬿：Co-IP assay to observe whether EBV Rta interacts with DNMTs. |
| 11/20/13 | 小嬿：Co-IP assay to observe whether EBV Rta interacts with DNMTs. |