小嬿

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condition2. 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

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 > 26b. Dox在2716的反應比TS佳, 但在26的反應比TS差c. 未比較protein expression level2. 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

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

To test Magnetic ChIP method:1. non-specific band was less than agarose method2. 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. 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

報告進度: 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 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 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

報告進度:1. TW01EREV_S無法用SAHA induction成功–>放棄S2. 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.

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.

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.