干货 | 免疫组化(IHC)实验从入门要高阶一篇搞定

干货 | 免疫组化(IHC)实验从入门要高阶一篇搞定

BOSTER 5天前

免疫组化(IHC)作为实验达人的必备技能之一,它的实验流程和方法并不难,但在实验过程中存在许多变量,容易遇到各种问题,因此,想要做出漂亮的实验结果并非易事,正所谓“细节决定成败”。下面小博将根据多年的实战经验为大家介绍下IHC的相关知识及在实验过程中经常遇到的问题和应对策略,从此,让成败与细节Say“拜拜”!

No.1

免疫组化(IHC)检测原理

免疫组织化学(immunohistochemistry,IHC)是一门利用酶、荧光素、生物素、重金属离子等作为示踪剂,通过免疫亲和(抗原抗体特异性结合),在组织切片或细胞薄片上原位显示追踪生物体内大分子物质动态变化规律的实用性染色技术。按其主要示踪原理可以分为免疫组化(默认酶标法)、免疫荧光法、胶体金法等。

作用:通过颜色来显示蛋白质的有无、定位(胞外、胞膜、胞浆、胞核)、含量变化

样本种类:

按来源:①组织;②培养细胞

按制作方式:①石蜡片(IHC-P)②冰冻片(IHC-F)③细胞片(ICC)

No.2

免疫组化(IHC)常见问题

无染色

原因
解决办法
一抗和二抗不匹配
使用针对一抗的二抗(如一抗来自兔,二抗为抗兔抗体)
没有足够的一抗与目标蛋白结合
提高一抗用量。延长4℃孵育时间(如过夜)
由于不当储存、稀释或反复冻融造成一抗/二抗试剂盒失效
做阳性对照确认一抗/二抗试剂盒的有效性
样本中没有目标蛋白
建议做阳性对照
目标蛋白含量太少
应用信号放大操作
脱蜡不彻底
延长脱蜡时间,更换二甲苯
固定液封闭了抗体识别表位
缩短固定时间,加强抗原修复
蛋白位于细胞核内(核蛋白),抗体不能穿透核膜
对样本进行破膜通透处理
PBS缓冲液被细菌污染后破坏了靶蛋白的磷酸根
在抗体PBS储存液中加入适量防腐剂,或使用新鲜无菌的PBS

高背景

原因
解决办法
封闭不充分
选择合适的封闭液,延长封闭时间
一抗浓度过高
针对一抗做浓度梯度实验,选择合适浓度
孵育温度过高,时间过长
选择4℃过夜或缩短孵育时间
二抗质量不佳
不加一抗,做二抗对照,选择合格二抗
组织冲洗不彻底
加强洗涤
内源性过氧化物酶含量过高
延长3%H2O2灭活时间或用0.5%高碘酸溶液室温孵育10min
固定过度
改变抗原修复方法或减小抗原修复强度。
信号过度放大
缩短抗体孵育时间
通透作用破坏膜并除去了膜蛋白
去除缓冲液中的通透剂
显色底物过量或显色时间太长
缩短底物孵育时间

非特异性染色

原因
解决办法
一抗/二抗浓度过高
降低抗体浓度和或缩短孵育时间
存在内源性过氧化物酶活性
延长3%H2O2灭活时间或用0.5%高碘酸溶液室温孵育10min
一抗与被染组织同源(如用鼠一抗检测鼠组织),加二抗后,二抗会与同源的所有组织结合
应用与组织非同源的一抗
切片/细胞变干
保持切片/细胞湿度,切勿变干。

No.3

免疫组化(IHC)实验流程

那么,怎样才能做出漂亮的染色结果呢?为了有一个全局概念,我们先来看一下石蜡切片免疫组化的实验流程吧!每一步都存在许多决定成败的小细节,它们可以是陷阱,也可以是获得好的实验结果的基石。

【免疫组化总流程图,点击查看大图】

01

取材如何操作

√ 快:尽量半小时内取材完成

√ 准:刀、剪锋利,一步到位轻夹轻放

√ 大小:1cm X 1cm X 0.2cm左右最宜

02

固定的注意事项

√ 何时固定?——取材后立刻!马上!

√ 用什么固定?——4%多聚甲醛。有致密外膜用Carnoy液,活检用Bouin液

√ 固定液用多少?——组织体积20倍以上最宜。量少应中途换液1-3次 

√ 固定多久?——6-24h最佳。固定越久所需修复强度越大,越容易出现自发荧光及非特异性染色。

03

选石蜡片还是选冰冻片?

√ 要保存简单 ——选石蜡片 !

√ 要速度快 ——选冰冻片!

√ 要结构漂亮 ——选石蜡片!

√ 抗原不稳定 ——选冰冻片!

√ 抗原稳定 ——石蜡片冰冻片皆可!

04

切片烤片如何操作?

√ 切多厚?

石蜡片3-8μm,一般5μm;冰冻片5-15μm,一般8μm

√ 怎么捞片?

水温40-45℃,组织平整后玻片有油漆的一面贴近组织向斜上方提起

√ 烤多久?

37℃过夜 或  60℃   1-2h

注意:使用防脱处理的玻片

05

脱蜡步骤

Step 1:二甲苯

Step 2:二甲苯

Step 3:二甲苯

Step 4:100%酒精

Step 5:85%酒精

Step 6:70%酒精

注:浸泡时间是每缸10min

06

06

灭活:3%H₂O2灭活做还是不做?

√ 内源性过氧化物酶在血管瘤、肝、胎盘、阑尾炎,坏死区域

和急性炎症组织含量较高

√ 显色系统为过氧化物酶(HRP)系统,该步骤建议一定要做

√ 碱性磷酸酶(AP)系统以及免疫荧光,该步骤可以不做

√ 在灭活时,如组织片中出现细小且密集的气泡,表示过氧化物酶含量过高,这时用3%H₂O2溶液难以完全阻断,可以用0.5%高碘酸溶液室温条件孵育10min

07

抗原修复

√ 抗原修复方式:酶修复、高压热修复、微波热修复,都能得到更好的效果

√ 消化酶的选择:需格外注意的各种消化酶的最佳工作PH值

√ 修复液的选择:柠檬酸盐缓冲液(PH6.0)和EDTA(PH8.0或9.0)修复液

经验证,对于大多数的抗体来说后者使用效果更优

√ 消化酶的选择:固定时间越久,修复强度越强,旧标本修复强度强于新鲜标本

08

封闭

√ 5%BSA是最常用

√ 血清,效果最全面

√ 封闭血清与二抗同源,与一抗不能同源

09

抗体孵育

√ 单抗和多抗各有优势,按需选用

√ 一抗4℃孵育效果最佳,备选37℃ 1-2h

√ 二抗需与一抗的种属、类别或亚类相匹配,推荐驴,山羊,绵羊等种属

√ 二抗/SABC 一般 37℃ 孵育30 min 

10

显色

√ DAB显色液现用现配

√ 建议镜下控制反应时间

√ 根据显色时间反向优化实验条件

11

复染/返蓝

√ Mayer`S 苏木素复染(不易过染)

√ 碱性溶液返蓝(如氨水、饱和磷酸氢二钠溶液等)

√ 免疫荧光用DAPI染核

12

脱水透明

Step 1:70%酒精

Step 2:85%酒精

Step 3:100%酒精

Step 4:二甲苯

Step 5:二甲苯

Step 6:二甲苯

13

封片

√ DAB显色用中性树胶封片

√ 免疫荧光建议用抗荧光衰减封片剂

√ AEC显色用水溶性封片剂

14

对照的设置

√ 阳性对照:主要用于验证实验流程有没有问题,各试剂质量是否过关,多用已有明确蛋白表达的组织

√ 阴性对照:确认染色结果的特异性,要用非相关抗体

√ 空白对照:用以排除非特异背景染色,一般多用一抗来源的动物血清

15

抗原定位

√ 胞膜型表达:

阳性染色颗粒主要定位于细胞膜表面。常见的有淋巴细胞膜抗原(如LCA、CD20、CD4、CD8等)、间皮细胞抗原(如MCA、CR、CK5/6等)、细胞膜受体(insulinR、FAS、CD25等)、黏附分子(整合素、干扰素、钙黏蛋白等)、上皮细胞抗原(EMA、ECA、ESA等)等。

√ 胞核型表达:

阳性颗粒定位于细胞核中。常见的有增殖细胞周期抗原(Ki-67、PCNA等)、性激素受体(ER、PR、AR等)、肿瘤相关基因(P53、P27、BRCA1等)、肌核抗原(Myf-3、MyoD1等)、淋巴细胞核抗原(OCT-2、MUM1等)、上皮细胞核抗原(P63、TTF-1等)等。

√ 胞质型表达:

阳性颗粒定位于细胞质。常见的有细胞结构蛋白 (中间丝蛋白、微丝蛋白等)、细胞内酶和功能蛋白(MPO、NSE、PLAP等)、神经内分泌物(GH、PLR等)等。

√ 胞膜-质型表达:

阳性颗粒分布于细胞质和细胞膜。常见的有肿瘤相关抗原(如CA家族抗原)、间皮标志物(MCA、CR等)等。

√ 胞核-质型表达:

阳性颗粒分布于细胞核和细胞质。常见的有S-100、HMB45、Melan A、HSP27、MLH1等。

16

数据库推荐

17

17

量化分析

√ 阳性着色细胞计数法:

在40*光镜下,随机选择不重叠的3-5个视野,人工或机器计数阳性着色细胞和总细胞数,比较阳性细胞比率

√ 灰密度分析法:

通过在不同组别和不同动物组织切片上选择相同区域相同条件下image j进行灰密度分析,然后进行统计分析即可

√ 评分法:

通过在光学显微镜下对组织切片分别按染色程度(0-3分为阴性着色、淡黄色、浅褐色、深褐色)、阳性范围进行评分(1-4分为0-25%、26-50%、51-75%、76-100%),最终可以分数相加,再进行比较。

免疫组化(IHC)中存在的细节性问题“千千万”,每一种都有可能影响实验的成败,以上篇幅限制,如果你还想更深入的学习IHC相关问题,可扫码观看我们前两期开展的《IHC/IF实验课堂》直播课,该课程由我们博士德非常资深的组化实验专家全面讲解,深度分析,帮助你轻松搞定免疫组化的“疑难杂症”!

Measure

Measure

点击数:0

样品比较珍惜?用trizol提取RNA时同时提取蛋白

亲自尝试了,效果ok。
蛋白溶解特别慢,特别慢,要有耐心。

具体的提取步骤如下:
1.
样品加氯仿分层后,移去上层水相(该水相里就是RNA,可照常继续进行RNA的提取),加0.3ml乙醇沉淀中间层和有机相中的DNA,涡旋混合,室温放置3分钟,2-8℃不超过2000×g离心5分钟。【注:这一步同时可以除去未完全消化的残渣】
2.
小心吸出沉淀后的上清,加1.5ml异丙醇沉淀蛋白质。室温放置10分钟,2-8℃12000×g离心10分钟弃上清。
3.
加2ml含0.3M盐酸胍的95%乙醇洗涤蛋白质沉淀。室温放置20分钟,2-8℃7500×g离心5分钟,弃上清,重复两次。【注:不太清楚为什么这个时候用这么强烈的变性剂?防止蛋白降解?】
4.
用2ml无水乙醇同样方法再洗一次。
5.
真空抽干蛋白质沉淀5-10分钟,用1%SDS溶解蛋白质,反复吸打,50℃温浴使其完全溶解,不溶物2-8℃10000×g离心10分钟除去。分离得到的蛋白质样品可用于Western Blot或-5至-20℃保存备用。【注:无水乙醇很容易挥发,所以几分钟就能抽干;没有真空抽干机也无所谓,把无水乙醇倒掉,开盖子静置几分钟就行了。这一步干燥千万不能太干了,否则溶解的时候很不爽——很难溶解.我是在50度水浴中过夜,或者每水浴30分钟就放在超声清洗机里清洗10分钟——虽然不是专门用来粉碎细胞的超声,但毕竟是超声波哦,还是蛮有效果的。:D】
注意事项:
1.以上各试剂的用量都是以1mLTrizol为准,Trizol体积变化,各试剂用量随之等比例变化。
2.蛋白质沉淀可保存在含0.3M盐酸胍的95%乙醇或无水乙醇中2-8℃一个月以上或-5至-20℃一年以上。
3.这一步可省去:用0.1% SDS在2-8℃透析三次,10000×g离心10分钟取上清即可用于Western Blot。

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冰冻切片免疫荧光_实验方法

市面上有不用冲洗的DAPI+抗淬灭剂出售,而且挺好用,这样省两个步骤,很香。

2.丙酮或多聚甲醛固定5-10min;
3.PBST洗5 minx3次;
4.(可选)抗原修复可选;
5.(可选)0.1% Triton-100X透膜;
4.加山羊血清封闭液或3%BSA封闭30min;
5.弃去封闭液,滴加相应抗体(1%BSA稀释),保湿盒中4℃过夜;
6.PBST洗 5 minx3次; 
7.滴加二抗,避光,37℃,1-2 h;
8.PBS洗 5 minx3次; 
9.DAPI染色 5 min; 
10.PBST洗 5 minx3次;
11.滴加抗淬灭封片剂并用盖玻片封片;
12.荧光显微镜或激光共聚焦显微镜下观察并拍照。

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大家有没有推荐使用的抗淬灭剂? – 生物科学 – 小木虫论坛-学术科研互动平台

大家有没有推荐使用的抗淬灭剂? 

问:推荐使用哪些抗荧光淬灭剂?
答:一些常用的和最有效的抗荧光淬灭剂的优缺点比较如下,也提供了部分的商业来源以及共聚焦显微镜用户的意见和评价。
1.p-phenylenediamine (PPD)
  尽管它是最有效的抗淬灭剂之一,但对光和热都有较强的敏感性,而且具有毒性,后者也限制了在体内研究的应用。Krenik 等(1989)建议最理想的 PPD 抗淬灭剂混合液配方是:90% 甘油、10% PBS,其中 PPD 的浓度在(2~7)mM 之间,最终pH值为 8.5~9.0。
2.n-propyl gallate (NPG)
  NPG 无毒性,对光和热稳定,但抗漂白效果不如 PPD,可用于体内研究。推荐浓度在(3~9)mM,用甘油配置效果也不错。配置的方法在FAQ的后部分。
3. 1,4-diazobicyclo[2,2,2]-octane (DABCO)
  DABCO是一种不电离的、稳定的抗淬灭剂,价格便宜而且容易使用。配置方法在 FAQ 的后部分。
4.Ascorbic acid (Vitamin C)
5.Vectashield
  公司描述:具有在显微镜观察中阻止荧光的丢失并在长期储存过程中保持抗荧光淬灭的能力。抑制荧光素的漂白,例如:Texas Red , Rhodamine , AMCA 和其他的荧光染料。特别的是,比甘油缓冲液、聚乙烯醇固定液或者其他含有普通抗荧光淬灭剂的固定液更加稳定,光学清晰度更高。
Vector Laboratories, Inc.
30 Ingold Road,
Burlingame, CA 94010 USA
Tel: (415) 697-3600
Fax: (415) 697-0339
6.SlowFade
  公司描述:SlowFade 的原始配方(S-2828)可将荧光素的荧光淬灭速率减小到零。尤其适用于激光扫描共聚焦显微镜的定量测定,因为其在测定时往往要求激发光强度较强,而且采集时间也较长。SlowFade 试剂可使荧光素的有效荧光发射扩大 50 倍以上,用该试剂封片后,细胞和组织中的荧光信号可保持两年之久。原始的 SlowFade 配方实际上可使荧光素的荧光完全淬灭,也可使 Cascade Blue 和 Alexa Fluor 350 荧光团的荧光几乎完全淬灭。
Molecular Probes, Inc.
P.O. Box 22010 Eugene, OR 97402-0414 USA
Tel: (503) 465-8300
FAX: (503) 344-6504
7.SlowFade Light
  为了克服上述的局限,分子探针公司的研究人员又研制了 SlowFade Light 抗荧光淬灭试剂盒。该抗荧光淬灭剂的配方使荧光素的淬灭速率降低 5 倍,而荧光素的初始荧光强度没有显著降低,因此使光学显微镜的信噪比显著提高。此外,对 Cascade Blue, Alexa Fluor 350, tetramethylrhodamine 和 Texas Red 的淬灭也达到最小。实际上,SlowFade Light 抗荧光淬灭剂试剂盒可将 Cascade Blue 的淬灭速率几乎降到零,而其发射强度仅降低约 30%。
问:DABCO抗荧光淬灭封片剂的配方是什么?
答:
DABCO 储藏液
1.在 60℃ 下,溶解 2 克 DABCO(抗荧光淬灭剂)于 90ml 甘油中,15-30 分钟。
2.加 10 ml 1M Tris-HCl,pH 7.5
3.用 5M HCl 调节 pH 值到 8.0
4.冷却到室温
5.加 100 ml 20% thimerosal(溶解于水)
6.可选:加 50 ml PI (储液:溶于水的 1 mg/ml PI )
7.4℃ 于棕色瓶或箔纸包裹的瓶子中避光保存
缩写:
PI = propidium iodide
PI – 激发光与发射光与罗丹明相似 – 用 488 nm 或者 514 nm 的激发光 – 长通 570 nm 或者 600 nm 的发射滤片
DABCO = 1,4-二偶氮双环[2,2,2]-辛烷 
Ordering (Sigma) :
D2522 DABCO, 25g
T5125 thimerosal, 10g
P4170 PI, 10mg
问:N -丙基没食子酸盐(n-propyl gallate ,NPG)抗荧光淬灭封片剂的配方是什么?
答:
溶于甘油的 5%N -丙基没食子酸盐
1.于 50 ml 试管中,加 5% N -丙基没食子酸盐到 25 ml 甘油中
2.摇晃混合过夜(室温或者 4℃)
3.使用之前静置一天,让溶液中的气泡跑掉
4.4℃ 于棕色瓶或箔纸包裹的瓶子中避光保存

Measure

Measure

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免疫荧光常规流程 | Cell Signaling Technology

免疫荧光法常规流程

封闭液还可以,以后可以都使用这种配方的封闭液。

一抗稀释液也可以,CST还是挺靠谱的。

A. 溶液和试剂

若想获得高质量的免疫荧光图片,请使用我们 #12727 Immunofluorescence Application Solutions Kit 中高效且划算的预制试剂。

:利用反渗透去离子水 (RODI) 或同等级别的水制备溶液。

  1. 20 X 磷酸盐缓冲液 (PBS):( #9808) 为了制备 1 L 1 X PBS:添加 50 ml 20 X PBS 至 950 ml dH2O,然后将其混匀。注:调节 pH 至 8.0。

  2. 甲醛:16%, 无甲醇,Polysciences 公司出品。(货号 18814),使用新鲜制剂,小瓶打开后避光 4°C 保存。按照 1 比 4 的比例在 1X PBS 中稀释,制成 4% 甲醛溶液。

  3. 封闭缓冲液:(1X PBS/5% 正常血清/0.3% Triton ™ X-100):准备 10 ml 缓冲液,添加 0.5 ml 与二抗同一物种来源的正常血清(例如加入 Normal Goat Serum (#5425) 到 9.5 ml 1X PBS 中)并混匀。搅拌时加入 30 µl Triton™ X-100。

  4. 抗体稀释缓冲液:(1X PBS/1% BSA/0.3% Triton™ X-100):准备 10 ml 溶液,添加 30 µl Triton™ X-100 到 10 ml 1X PBS 中。混匀后再加入 0.1g BSA (#9998),并混匀。

  5. 荧光物质标记二抗:(抗小鼠 #4408, #4409, #8890, #4410) (抗兔 #4412, #4413, #8889, #4414)(抗大鼠 #4416, #4417, #4418)。

  6. Prolong® Gold Antifade Reagent (#9071), Prolong® Gold Antifade Reagent with DAPI (#8961)。

B. 标本准备

I. 培养细胞系 (IF-IC)

:细胞应当在多孔板、腔室玻片或盖玻片上直接培养、处理、固定和染色。

  1. 吸干液体,随后在细胞上覆盖一层 2–3 mm 用温热 PBS 稀释的 4% 甲醛。

    :甲醛有毒性,仅限通风橱中使用。

  2. 室温下固定细胞 15 分钟。

  3. 吸干固定液,用 1X PBS 漂洗三次,每次 5 分钟。

  4. 继续进行免疫染色操作(C 部分)。

II. 冰冻/低温切片 (IF-F)

  1. 冰冻固定组织进行免疫染色(C 部分)。

  2. 对新鲜未固定的冰冻组织,立刻按下列步骤固定:

    1. 将切片覆盖一层用温热 1X PBS 稀释的 4% 甲醛溶液。

    2. 室温下固定切片 15 分钟。

    3. 用 PBS 漂洗玻片三次,每次 5 分钟。

    4. 继续进行免疫染色操作(C 部分)。

C. 免疫染色

:所有随后的孵育都应当在室温下完成,除非另外注明需要在潮湿光密盒或带盖的培养皿/板中孵育,以防止干燥和荧光物质淬灭。

  1. 在封闭缓冲液中封闭标本 60 分钟。

  2. 封闭标本时,按照数据表中推荐的稀释比例,在抗体稀释缓冲液中配制一抗。

  3. 吸去封闭缓冲液,加入稀释后的一抗。

  4. 4°C 孵育过夜。

  5. 用 1X PBS 漂洗三次,每次 5 分钟。

    :如果使用荧光物质标记的一抗,则跳转到 C部分,第 8 步。

  6. 用抗体稀释缓冲液将荧光物质标记的二抗稀释后,室温下避光孵育标本 1–2 小时。

  7. 用 1X PBS 漂洗三次,每次 5 分钟。

  8. 使用 Prolong® Gold Antifade Reagent (#9071) 或 Prolong® Gold Antifade Reagent with DAPI (#8961) ,用盖玻片盖住切片。

  9. 要达到最佳效果,使用封片液室温过夜。将切片平放避光 4°C 环境下,可长期保存。

发布于 2006 年 11 月 

修订于 2016 年 7 月

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Comparative Analysis of Cas9 Activators Across Multiple Species

Comparative Analysis of Cas9 Activators Across Multiple Species

The publisher’s final edited version of this article is available at Nat Methods

See other articles in PMC that cite the published article.

Associated Data

Supplementary Materials

Abstract

Several groups have generated programmable transcription factors based on the versatile Cas9 protein, yet their relative potency and effectiveness across various cell types and species remain unexplored. Here, we compare Cas9 activator systems and examine their ability to induce robust gene expression in several human, mouse, and fly cell lines. We also explore the potential for improved activation through the combination of the most potent activator systems and assess the role of cooperativity in maximizing gene expression.

Introduction

Cas9 is an RNA-guided endonuclease that can be directed to cut a selected site1,2. This process requires complementarity between the Cas9-associated guide RNA (gRNA) and the target site, along with the presence of a short protospacer adjacent motif3,4,5,6. Early efforts at engineering the Cas9 protein uncovered several residues involved in DNA catalysis that, when mutated, generate forms of the protein that are still capable of DNA binding but lack detectable nuclease activity4,7,8. These nuclease-null, or “dead,” Cas9 (dCas9) variants can then be fused to effector domains, allowing users to precisely direct a given functional activity to any arbitrary locus within the genome7,9,10,11. Recently, several groups have generated systems to endow dCas9 with the ability to activate gene expression, with dCas9-VP64 representing the first activator and the standard against which subsequent “second-generation” activators are typically compared12,13,14,15,16,17,18,19. Due to differences in the cellular context in which the various second-generation activators were tested and non-uniformity with regard to the particular target genes, guide RNA, transfection conditions, and time to analysis of gene induction, it remains ambiguous which system is the most potent and whether any individual system possesses unique properties not displayed by the others20,21,22. Here, we conduct a survey of the various second-generation activators, identifying the most potent systems, which we rigorously characterize across a plethora of target genes and species. These data confer much needed guidance to those wishing to adopt dCas9 activator technology and provide the community with an extensive set of validated reagents to aid the adoption of these tools within labs without previous experience.

Results

The number of second-generation dCas9 activators is too large to be systematically tested across a large panel of target genes and cell lines. Accordingly, we first performed a series of pilot experiments within human embryonic kidney (HEK) 293T cells to compare representative examples of all published dCas9 activators (Fig 1a). As expected, for the two target genes tested, the majority of second-generation systems show improved levels of activation as compared to dCas9-VP64, with three activators in particular – VPR, SAM, and Suntag – appearing to be the most potent (Fig 1b and activators described in detail in Supplementary Note 1)12,13,15,16. Based on these initial data, we decided to focus our efforts on VPR, SAM, and Suntag.

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Figure 1

Initial tests of all second-generation activators on endogenous genes in HEK293T cells

(a)dCas9-VP64 and dCas9-VPR both work via activation domains fused to the C-terminus of Cas9. SAM uses dCas9-VP64, but recruits more activation domains to the gRNA. Scaffold recruits multiple copies of VP64 to the gRNA. Suntag uses single chain antibodies to recruit multiple copies of VP64 to the peptide tail. P300 uses the catalytic core of the epigenetic modifier fused to dCas9 to modify the chromatin around the promoter to drive transcription. VP160 is the direct fusion of 10 repeats of VP16 protein to dCas9 instead of the usual four that makes up VP64. VP64-dCas9-BFP-VP64 drives transcription via the fusion of VP64 to both the N and C-termini of Cas9 (b)Data indicate the mean + s.e.m (n = 2 independent transfections).

When compared across a panel of coding and non-coding genes, VPR, SAM, and Suntag demonstrate the ability to induce potent gene expression. At times, this activation reached levels several orders of magnitude higher than the first-generation dCas9-VP64 activator (Fig. 2a and Supplementary Fig. 1). SAM was the most consistent in delivering high levels of gene induction, although it always remained within five-fold of either Suntag or VPR, neither of which was generally superior to the other (Fig. 2a). Previously, a negative correlation between the basal expression state of a given gene and the fold change in gene expression upon targeted upregulation by dCas9-based activators was reported12,13. In other words, lowly expressed genes tend to have a higher fold induction than highly expressed genes. We find that this phenomena is a general principle for each of the tested synthetic activators, which suggests that these systems are only capable of inducing gene expression to some static upper limit, dependent upon the activator architecture employed (Supplementary Figure 2).

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Figure 2

Activation of endogenous genes in HEK 293T cells

(a) RNA expression analysis on 6 endogenous human genes. Data indicate the mean + s.e.m (n = 2 independent transfections). (b) Multiplexed activation of six endogenous human genes. Data indicate the mean + s.e.m (n = 2 independent transfections).

One of the benefits of dCas9-based transcriptional effectors over other synthetic transcription factors is the ease by which multiple loci can be upregulated, only requiring the provision of an extra gRNA for each additional locus one desires to activate. Previous work has shown a general decrease in gene activation upon multiplexing, but whether this is true of all systems is unknown12,19. To address this question, we performed a series of multiplex activation experiments (Supplementary Fig. 3). When activating three genes at once between VPR, SAM, and Suntag, no system appeared superior to the others with all showing similar levels of activation, within an order of magnitude of each other for the majority of target genes. This is a somewhat surprising finding given that SAM and Suntag are believed to require the assembly of large multimember protein complexes in order to generate a highly competent activator. To further investigate the limitations of multiplexing, we simultaneously directed each of the systems to six target genes at once. Even within this more complex activation scheme all systems showed similar levels of relative gene activation, and in doing so highlight the robustness of these tools to actuate complex transcriptional regulation (Fig 2b). Of note, we observe within our control samples inherent variation in the level of basal gene expression upon which we base our calculation of relative fold induction. We are therefore unable to make comparisons with regard to the absolute amount of activation a given system can perform when targeted to a single gene versus multiple genes by comparing expression data across experiments.

An important concern for any Cas9-based technology is the specificity of the desired effect. It has been demonstrated that Cas9 binds promiscuously throughout the genome23. This effect could potentially result in aberrant transcription for Cas9-activators if they were to bind to the wrong promoter. To test the specificity of each activator, we performed whole RNA-seq on samples with each of our activators targeting HBG1 (Figure 3). We found that the correlation in gene expression between each activator and our control sample was very similar to the correlation between biological replicates in our dataset (R ~ 0.98 in each case), indicating that gene expression is not broadly affected by the presence of any activator (Figure 3a). Disregarding noise from genes with low baseline expression (<0.1 TPM), HBG1 was the most highly upregulated gene in each sample except for VP64, indicating that the activators are highly specific (Figure 3b).

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Figure 3

Evaluation of activator specificity by RNA sequencing

(a) Gene expression levels (log2TPM, Transcripts Per Million) in cells transfected with the indicated activator targeting HBG1 (y axis) vs. expression in cells transfected with guide RNA only (x axis). R indicates Pearson’s correlation coefficient, calculated for log-transformed values on all genes except HBG1. Genes with 0 TPM in both replicates of either the activator or control were excluded before log transformation. Average of two biological replicates shown. (b) Histograms showing the distribution of fold-changes in gene expression (activator/guide control). Genes were filtered to include only those with TPM > 0.1. Bottom panel is zoomed in on the range 2-400. In both panels arrows indicate fold-change of HBG1 for the indicated activator. Average of two biological replicates shown.

To further explore the generality of the trends we observed within HEK293T cells, we targeted a smaller subset of genes within other biomedically relevant human cell lines (Hela, U-2 OS, and MCF7). While Hela cells demonstrated a similar trend of SAM leading to more potent activation, U-2 OS and MCF7 cells showed a deviation from this trend with Suntag and VPR being the more potent activators (Fig 4a and Supplementary Fig 4a). The biological basis for these cell-line specific differences remains to be determined and is not likely due to differences in basal expression state given that HBG1 and TTN are expressed at a similarly low basal state across the tested cell lines (data not shown).

Open in a separate window

Figure 4

Activation of endogenous genes in alternative human, mouse and fly cell lines and effects of multiple guides

(a) Activation of endogenous genes in Hela, U-2 OS and MCF7 cells. Data indicate the mean + s.e.m (n = 2 independent transfections). (b) Activation of endogenous genes in mouse and fly. Data indicate the mean + s.e.m (n = 2 independent transfections) (c) Samples were transfected with the indicated guide or mixtures of guides. Theoretical sum indicates sum of the relative RNA expression for each activator of the individual gRNAs. Data indicate mean +S.E.M. (n = 2 independent transfections)

Because human cell lines represent only a single context within which dCas9-based activators might be employed, we performed a series of experiments within mouse N2A and 3T3 cells as well as Drosophila S2R+ cells13. As a whole, all second-generation activators showed improved activity as compared to the dCas9-VP64 standard. Within mouse and Drosophila cells, VPR, SAM, and Suntag showed similar levels of gene induction (within five-fold of each other), with the most potent activator changing depending on the target analyzed (Fig 4b and Supplementary Fig 4b).

Given that VPR, SAM, and Suntag represent divergent approaches to Cas9 activator design, we sought to determine if any of the activator components could be combined to generate a hybrid activator with increased potency. First, we naively combined each of the systems (Supplementary Fig. 5). For example, we used the Suntag dCas9 component and the SAM-modified gRNA to simultaneously recruit both scFv-VP64 to the end of dCas9 and p65-hsf1 to the gRNA. In addition, we tested if systems such as SAM or Suntag that rely on recruiting several copies of their activator p65-hsf1 or VP64, respectively might be enhanced by nucleating a different activator such as VPR (Supplementary Fig. 6). Finally, we investigated if the MS2 hairpins utilized within the SAM system to recruit the MS2 binding protein-p65-hsf1 activator component might be enhanced by combining them in part or in whole with the scaffold method of MS2 binding protein mediated activator recruitment (Supplementary Fig. 7). Towards this goal a hybrid SAM+Scaffold gRNA was generated along with a series of chimeras containing select portions of each system combined together. Despite our exhaustive attempts, there was no combination of dCas9 activators, secondary effector component alteration, or gRNA modification that could lead to a chimeric system with enhanced transcriptional regulatory capacity for either single or multiplex gene induction.

Previous work with the first-generation dCas9-VP64 activator uncovered a role for cooperativity in enabling synergistic increases in gene activation7,10,11,24,25. Whether this observation is also true for the significantly more potent second-generation activators remains unexplored. It is likely that the extendibility of these newer systems may be limited from further increasing gene expression due to insufficient host resources (such as local RNA polymerase concentrations near a given locus)26. To study the effects of recruiting multiple dCas9 activator complexes to the same locus, we generated three gRNAs against a lowly, intermediately, and highly expressed gene target (ASCL1, NEUROD1, and CXCR4 respectively) within HEK293T cells and transfected each of the gRNAs alone or all the gRNAs against a given target gene at once. Simultaneous targeting of the same gene with multiple gRNAs led to additive or greater levels of gene activation (Fig. 4c).

Discussion

Cas9-based activation represents a powerful method to study gain-of-function phenomena on genomic scales and has already been used to uncover mechanisms of toxin-mediated cell death and resistance of tumor cells to targeted therapy.12,15 While it is tempting for those analyzing the initial Cas9 activator publications to make cross-study comparisons, these types of analysis are fraught with confounding variables, such as differences in the cell line used, gRNA selected, amount of Cas9 complex transfected, and time until RNA extraction. In addition, often overlooked variation in the basal expression level of some of the more lowly expressed target genes can lead to large differences in the perceived performance of one system over another. Within our own data, there are instances where the absolute level of target gene activation differs due to these changes, despite these experiments being performed under similar conditions (Fig 1 and Supplementary Fig 1). This source of variation emphasizes the need for comparisons across activators to be performed within a single experiment if users want to make statements with regard to absolute levels of gene induction. In contrast, for cases where understanding a relative difference in performance is the primary goal, differences in basal expression state will no longer play a role as each activator is compared to each other thus eliminating variance due to basal expression state.

Our results demonstrate that, across a range of target genes and cellular environments, the VPR, SAM, and Suntag systems are consistently superior to the previous VP64 standard. In addition, while SAM shows a trend for more potent activation in some contexts, VPR, SAM, and Suntag generally fall within an order of magnitude of each other with regard to fold increase in gene expression. It is additionally comforting that each activator was similarly specific. This suggests that constraints outside of the ability to induce gene expression or specificity, such as ease of delivery, familiarity with the system, and access to the necessary reagents, may be more practical concerns to consider27.

It is assuring that the second-generation activators show robust activation even when employed in organisms such as Drosophila that are very distantly related to the human cell lines in which these tools were developed. The demonstrated portability of these dCas9 activators across disparate model systems and species suggests that they hold potential as universal genetic tools. It is likely that these systems will prove effective across a wide range of scientifically interesting organisms with minimal additional engineering.

Our attempts to build an improved chimeric activator by fusing elements from VPR, SAM, and Suntag were unsuccessful (Supplementary Figs. 5-7). This is particularly unexpected given the drastically different ways in which the systems generate a functional activator1216. It is difficult to disentangle issues such as steric interference between neighboring activator elements from the possibility that each of these systems28, while different, may be interacting with a similar subset of the transcriptional regulatory machinery29,30. Future efforts to improve dCas9-based activators may benefit from exploring other unique architectures or novel activation domains, along with examining the use of epigenetic modifiers to provide complementary mechanisms to further enhance activation18,31.

While we did not perform experiments to explicitly address potential differences in gRNA binding site preferences between VPR, SAM, and Suntag, it is telling that for experiments where several gRNAs against the same gene were examined, the activators show the same gRNAs as being either the most potent or the least potent (Fig. 4c). These data suggest that the rules governing activator placement within a given promoter region are shared. It will be interesting to characterize the properties shared by highly potent gRNAs to help distinguish effects of distance from the transcriptional start site from features such as gRNA sequence composition and stability.12,15,32,33,23 In addition, the fact that we are able to achieve increased levels of programmable gene induction by employing multiple gRNAs against a single target gene suggests that none of the current Cas9 activators have yet to achieve “maximal activation”. Since if they were, each activator would not have been aided by the use of multiple guides. These results suggest that continued improvements to Cas9 activators are still possible and should aid in generating a more reliable tool for performing systematic genome-wide screens.

Although not highlighted in any of the original studies for VPR, SAM, or Suntag, our results suggest that the combination of multiple gRNAs represents a viable strategy to enhance gene expression in cases where maximal induction is desired. This strategy may be most valuable for highly expressed genes, which are generally recalcitrant to large amounts of overexpression from the native locus12,13. It could also be used to assure that all genes are consistently upregulated given that even for easily upregulated genes there can be over a hundred-fold difference in potency between various gRNAs (Fig. 4c).

Materials and methods

Vector design and construction

Vectors used and guide scaffolds will be deposited to Addgene

dCas9-VP64 (Addgene #47319), dCas9-VPR (Addgene #63798), dCas9-VP64 (for SAM activation Addgene # 61425), MCP-p65-hsf1 (Addgene #61426), dCas9-10xGCN4 (Addgene #60903), scFv-VP64 (Addgene #60904), dCas9-p300core (Addgene #61357), dCas9-VP160 (Addgene #48225), Cas9-m4 (Addgene #47316) and MCP-VP647 were previously described.

For all systems tested the original vectors deposited to Addgene were employed allowing us to use the presumably optimal expression vector decided upon by the various depositing laboratories. dCas9-VP64, dCas9-VPR, dCas9-p300, dCas9-VP160, and dCas9-m4 constructs were all driven under CMV promoters. dCas9-VP64 (for SAM activation) and MCP-p65-hsf1 used EF1alpha promoters and dCas9-10xGCN4 and scFv-VP64 used SV40 promoters.

Gibson cloning was utilized to make all variants. For SAM and Scaffold gRNA variants, gene blocks were used (Integrated DNA Technologies). See Supplementary Note 2 for full sequence information.

All dCas9 activator components were cloned into a Drosophila pActin vector using Gibson assembly,34,35 adding a Kozak sequence (GCCACC) immediately upstream of the start codon.

To generate Drosophila MS2-containing sgRNA expression vectors for SAM and Scaffold, the pCFD3 plasmid was modified via Gibson assembly to include the indicated sgRNA tails.36 For all sgRNA plasmids guide oligos were cloned using a BbsI digest.36

Mammalian cell culture

All human and mouse cell lines were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS and penicillin-streptomycin (all from ThermoFisher) in an incubator set at 5% CO2 and 37° celsius. MCF7 (gift of J. Lee, Cold Spring Harbor, Cold Spring Harbor, NY) media was supplemented with 0.01mg/ml human recombinant insulin (EMD Millipore). HEK293Ts, Helas (both gifts of P. Mali, UCSD, San Diego, CA), U-2 OS (ATCC HTB-96), MCF7s, N2As (ATCC CCL-131), and NIH-3T3s (gift of S. Shipman, Harvard Medical School, Boston, MA) were seeded into 24-well plates for transfection.

Cell lines were not tested for authenticity and were tested yearly for mycoplasma.

Mammalian transfections

DNA was aliquoted into individual tubes prior to transfection. 25ng of plasmid DNA containing the Cas9 component and 10ng of plasmid DNA with the guide was transfected into each well. For activators requiring a second component (Suntag, SAM, Scaffold), 100ng of plasmid DNA containing that component was also transfected. For all the other activators, 100ng of an empty vector (Puc19) was substituted for the secondary component in order to ensure equal amounts of DNA were transfected. For experiments involving multiple guides targeted towards one gene, the amount of gRNA plasmid per transfection was scaled so that the total plasmid gRNA in each well was 10ng. For example, for three gRNAs activating one gene, 3.33ng of each guide was transfected. In experiments involving multiple guides targeted towards different genes, 10ng of gRNA plasmid DNA was transfected per target. For all gRNA spacer sequences, please see Supplementary Table 1.

293T cells were transfected with Lipofectamine 2000 (ThermoFisher) using the following protocol. For each transfection, a stock solution of 50 μl of Opti-Mem (ThermoFisher) and 2 μl of Lipofectamine 2000 per transfection was made. The solution was then vortexed and incubated for five minutes. The Opti-mem/Lipofectamine solution was then added to the individual aliquots of DNA, vortexed and spun in a centrifuge at 100RPM for 1 minute. The DNA was then incubated for 30 minutes before being added to the cells.

All other cell lines were transfected with Lipofectamine 3000 (ThermoFisher) using the following protocol. 25 μl of Opti-MEM was added to each DNA aliquot along with 1 μl P3000 reagent per transfection. Master mixes of Opti-MEM and Lipofectamine 3000 were prepared with 25 μl of Opti-Mem and 1 μl or 0.5 μl of Lipofectamine 3000 per transfection. MCF7s, N2As, and U-2 OSes were transfected using 1 μl of P3000 reagent and 0.5 μl of Lipofectamine 3000 reagent per transfection. N2As and NIH-3T3s were transfected with 1 μl of P3000 reagent and 1 μl of Lipofectamine 3000 reagent per transfection. The solutions were vortexed and incubated separately for five minutes before 25 μl of the Lipofectamine 3000/Opti-Mem mix per transfection were added to the DNA aliquots. These new solutions were then vortexed, centrifuged at 100RPM for 1 minute, incubated for 30 minutes and then added to the cells.

To avoid excess toxicity to the cells, media was changed after 24 hours for the 293T, N2A and NIH-3T3 cells. Otherwise, media was changed after 4 hours.

RNA extraction and qPCR analysis for mammalian cell lines

Cells were harvested for RNA 48 hours post-transfection. RNA was extracted using the RNeasy Plus Mini Kit (Qiagen). cDNA was synthesized using the iScript cDNA synthesis kit (Bio-Rad) using 500ng of RNA per cDNA reaction. qPCR reactions were prepared using the KAPA SYBR Fast Universal 2x quantitative PCR kit using 0.5 μl of cDNA per reaction in a 20 μl total reaction volume. Gene expression was normalized to the expression of the gene ACTB for human cell lines and the mouse ortholog (actb) for mouse cell lines as an internal housekeeping gene. Cycling conditions were as follows: 95° for 1 minute, 95° for 10 seconds and 60° for 30 seconds. The latter two steps cycled for 50 repeats with plate reads taken after the 60° step. If a sample failed to amplify after 50 cycles, an arbitrary Cq count of 50 was substituted (this occurred in a single instance). For qPCR primer sequences, please see Supplementary Table 2.

RNA-seq analysis

For each sample, 100 ng of total RNA was DNase treated with Turbo DNase (ThermoFisher Scientific) at 37C for 30 minutes and then cleaned up with Agenocourt RNAClean XP Beads (Beckman Coulter). The RNA samples were polyA selected using Dynabeads mRNA Direct Purification Kit (ThermoFisher Scientific). RNA-Seq Libraries were constructed using Maxima H Minus First Strand Synthesis Kit (ThermoFisher Scientific) with random hexamers and then the NEBNext mRNA Second Strand Synthesis Module (New England Biolabs). The resulting cDNA was cleaned up with Agencourt AMPure XP Beads (Beckman Coulter) and then went into the Nextera XT DNA Library Prep Kit (Illumina). Final Libraries were once again cleaned with Agencourt AMPure XP Beads and analyzed on a BioAnalyzer using a High Sensitivity DNA Analysis Kit (Agilent). Libraries were quantified using a Qubit dsDNA HS Assay Kit (ThermoFisher Scientific), pooled, and and run on one lane of an Illumina HiSeq 2500 using 2×25 bp paired end reads. Reads were aligned to the hg19 UCSC Known Genes annotations using RSEM v1.2.1 and were analyzed in Python37 Histograms showing the distribution of fold-changes in gene expression (activator/guide control). Genes were filtered to include only those with TPM > 0.000001. Arrows indicate fold change of HBG1 for the indicated activator. Average of two biological replicates shown. Differential gene-expression analysis was done using the Voom38 and Limma39 packages in R for all genes with ≥0.000001. TPM mapped reads in each replicate, where differential expression was defined by a Benjamini-Hochberg adjusted P value of <0.05 and fold change of >10 or <0.1.”

Drosophila melanogaster transfections

S2R+ cells were transfected in 24-well plates using Effectene reagent (Qiagen). Each well was transfected with 50 ng of dCas9 component, 200 ng of any additional activator component, and 15 ng of sgRNA, and empty vector was used to equalize the total amount of DNA in each transfection to 265 ng. For multiplex reactions, 15 ng of each sgRNA were added.

Total RNA was collected three days after transfection, and qPCR was conducted as previously described.40

Supplementary Material

1

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2

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Acknowledgements

We would like to thank S. Vora, A. Tung, M.K. Cromer, and all the members of the Church and Collins lab for helpful discussions and technical assistance. G.C. acknowledges support from the US National Institutes of Health National Human Genome Research Institute grant P50 HG005550, and the Wyss Institute for Biologically Inspired Engineering. In addition, A.C.was funded by the National Cancer Institute grant 5T32CA009216-34, R.C. was funded by a Banting postdoctoral fellowship from the Canadian Institutes of Health Research and J.J.C. was supported by the Defense Threat Reduction Agency grant HDTRA1-14-1-0006. B.E.-C. acknowledges funding from the National Institutes of Health (NIH) under the Ruth L. Kirschstein National Research Service Award F32GM113395 from the NIH General Medical Sciences Division. We would also like to thank J. Lee, P. Mali, and S. Shipman for gifting us cell lines.

Footnotes

Contributions: A.C. and M.T. conceived of the study. A.C., M.T., B.W.P., and R.C. designed and performed experiments. S.J.H., R.J.C., and J.B. performed experiments.B.E.C, B.E.H and N.P. designed and performed all experiments in Drosophila Melanogaster. D.T-O and E.J.K.K. performed RNA-seq experiments and analyzed data. J.J.C and G.M.C. supervised the study. A.C. and M.T. wrote the manuscript with support from all authors.

Conflict of interest: G.M.C. has equity in Editas and Caribou Biosciences.

Accession codes:

Primary accessions:

Gene Expression Omnibus

{“type”:”entrez-geo”,”attrs”:{“text”:”GSE80611″,”term_id”:”80611″}}GSE80611

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学术争鸣 | 从迷雾重重到柳暗花明?全面起底DNA 6mA修饰相关研究的争议

学术争鸣 | 从迷雾重重到柳暗花明?全面起底DNA 6mA修饰相关研究的争议

编者按:围绕DNA新修饰6mA的争议巨大,BioArt编辑部在广泛听取了十多位海内外专家的意见的基础上专门对整个研究历程以及争议进行了回顾和描述,本着对科学负责的态度,BioArt编辑部保持了最大的独立性完成此文,全文8000多字,历时一年左右完成。尽管争议还在继续,但是相信这篇文章对关心和希望了解6mA领域的读者有一定的帮助,至于是非曲直,留给读者去思考和判断。

撰文 | 小柚

责编丨丁广进

DNA甲基化是最早被发现,也是目前研究最深入的表观遗传调控机制之一。哺乳动物中最主要的DNA修饰是5mC(5-甲基胞嘧啶),占人类DNA中总胞嘧啶的3%~6%【1】。相反,5mC在原核生物中很少,而6mA(N6-甲基腺嘌呤,对应RNA上的修饰成为m6A,请读者注意区分)则是原核生物中最具代表性的DNA修饰,主要参与限制-修饰系统(restriction-modification),保护个体免受外来DNA的侵入。

尽管哺乳动物中缺乏已知的限制-修饰系统,但近年来的研究在真核生物,甚至包括哺乳动物和植物基因组中鉴定到了6mA,并发现6mA在生长发育和疾病调控中具有重要作用。这些研究掀开了真核生物表观遗传修饰的新篇章。

但同时,随着关注的增加,不同的声音也不断被发出,一场围绕真核生物中DNA 6mA修饰的相关争议早已拉开帷幕,而最为激烈的是关于哺乳动物基因组中是否存在6mA的争议。

一、 “繁花似锦”——真核生物中6mA的鉴定历程

2015年,Cell连发3篇研究(入选当期Cell杂志的封面论文),首次报道藻类【2】、果蝇【3】和线虫【4】的基因组中具有6mA修饰, 并配同期评论“An Adenine Code for DNA: A Second Life for N6-Methyladenine【5】”,认为真核生物中6mA的发现,将赋予6mA研究的第二次生命。

Cell杂志的封面。Image design by Lena Kay.

仅过去一年,2016年Nature发表研究报道6mA在小鼠胚胎干细胞中存在,并参与转座子活性的调控,同时鉴定了ALKBH1为6mA去甲基化酶【6】

6mA在真核生物中的鉴定研究,不少论文登上Top期刊,足以说明6mA带来的惊喜和受到的巨大关注。

随后,越来越多的研究团队加入6mA研究。

2018年7月,Molecular Cell发表利用SMRT测序首次获得人类细胞的DNA 6mA修饰图谱的研究,证明6mA在人类基因组(包括线粒体基因组)中广泛存在,并且癌组织中6mA的丰度显著低于正常组织,同时鉴定了N6AMT1为6mA甲基化酶【7】。(详见BioArt报道:

)。

4个月后,Cell发表6mA在神经胶质瘤中的研究,发现6mA在脑癌干细胞中的含量高于正常神经组织,并且靶向6mA去甲基化酶ALKBH1有望成为治疗该类癌症的新策略(详见BioArt报道:

【8】

同时,植物6mA研究也在如火如荼的进行,势头不亚于6mA在人类中的研究。2017年,水稻和拟南芥基因组中6mA修饰图谱于2018年分别发表在Nature Plant【9】Molecular Plant【10】Development Cell【11】

不仅科学研究者们看到了6mA的潜力,多家测序公司也嗅到商机,声称可开展6mA测序鉴定服务,绘制特定体系中的6mA图谱 。

参考RNA m6A被证明广泛存在于mRNA并参与多项生命活动进程后的火热程度,许多研究同行预见DNA 6mA可能将成为表观遗传领域的热门新领域!

二、“针锋相对”——6mA的鉴定争议

虽然6mA在高等真核生物中的研究看起来花团锦簇,但我们仍然难以绘制6mA分布图谱和了解它的生物学意义,这是因为不同研究中6mA的丰度,分布和功能在有巨大差异。在植物和真菌中,6mA主要富集在基因转录起始区,与转录激活有关,丰度在0.05%至2.8%【2,11】。在脊椎动物中,6mA主要富集在转座子区【6】、异染色质区【8】甚至基因编码区【7】;而6mA的丰度在不同哺乳动物和组织中竟相差10000倍(0.0001%~1%)【7, 8,12-14】

这些差异,特别是6mA的丰度差异,是由于物种或组织特异性造成的?还是由于实验手段和方法的差异造成的?当6mA的丰度为0.0001%时,相当于每百万个腺嘌呤核苷酸(A)仅有10个携带6mA修饰,这样低的丰度,真的具有生物学功能吗?是否是检测时的污染?

关于真核生物是否真的具有6mA修饰,学界一直存有争议。

Schiffers SRatel D等分别在Angew Chem Int Ed Engl【9】FEBS Lett【10】发表研究直言小鼠中不存在6mA修饰。但这两项研究并未引起太大关注。

2019年6月,波士顿儿童医院的Eric L. Greer 博士在BMC Genomics发表研究 “Sources of artifact in measurements of 6mA and 4mC abundance in eukaryotic genomic DNA”,该研究发现由于检测技术中存在污染,目前大部分研究都高估了真核生物中6mA和4mC的含量

质谱检测(UHPLC-MS/MS,ultra-high performance liquid chromatography tandem-mass spectrometry)是各类修饰鉴定的金标准。在DNA 6mA的质谱检测中,需要使用核酸酶和磷酸酶将待检测DNA消化成单个脱氧核苷酸分子,而目前使用最广泛的核酸酶和磷酸酶都来源于细菌。细菌中含有高丰度的6mA,这可能使这些酶携带6mA。

确实,研究者检测了目前市面上3款常见的核酸酶和磷酸酶:Nuclease P1,Nuclease S1和DNA degradase plus,发现它们均含有6mA修饰的污染。当采用这些酶处理的水作为对照,并将其中6mA含量设置为背景值时,研究者发现包括线虫(这也提示研究人员,基于线虫体系围绕6mA开展的进一步深入研究所得出的结论【19】也需要重新审视),小鼠和人在内的多个物种,6mA含量非常低,甚至检测不到(< 0.00003% for 6 mA)。

这说明6mA在某些哺乳动物中的高丰度,是由细菌污染造成的;当排除细菌污染后,6mA在哺乳动物中的含量非常非常低。(值得注意的是,该研究的通讯作者Eric L. Greer之前是施扬教授的博士后,也是2015年在Cell线虫中6mA鉴定研究的第一作者。虽然BMC Genomics杂志的影响因子不高,但该研究的作者中有施扬和何川两位领军人物的名字,足以说明他们对该研究提及的细菌污染问题的重视。)

此外,今年3月,来自瑞典林雪平大学的Colm E. Nestor教授在Science Advance发表研究“No evidence for DNA N6-methyladenine in mammals”,通过多种检测方法,严格的对照设置和分析,对6mA在基因组中的存在进行了全方位否定。基于6mA抗体的Dot blot和DIP-seq,质谱和基于测序方法的SMRTseq(根据甲基化修饰会影响DNA合成速度间接反应6mA的含量)是6mA检测的最常用方法。

其中,Dot blot和DIP-seq的准确性主要依赖6mA抗体的特异性。研究者采用全基因组扩增DNA(whole genome amplification DNA,WGA DNA,经PCR产生,无6mA修饰)作为阴性对照,检测了多种组织和细胞系中6mA的含量。经检测,它们的6mA Dot blot强度与WGA DNA(阴性对照)相当,提示6mA的含量很低。

研究者进一步分析了已发表的6mA位点和丰度研究,发现即使是相同的组织或细胞,DIP-seq和SMRTseq检测到的6MA位点仅1~8%重合,质谱检测到的6mA含量更是相差10~1000倍,说明这些6mA的差异是课题组的不同造成的,而不是其本身的差异。总的来说,该研究认为迄今发表的研究无法支持哺乳动物基因组中存在6mA修饰或者有高丰度。

三、 “峰回路转”——6mA在哺乳动物基因组中存在,但却不是表观遗传标记(epigenetic mark)

6mA在人类基因组中的丰度差异,促使研究者们开发更精确,稳定和无污染的质谱方法鉴定6mA的含量。

今年3月,来自德国IMB的Christof Niehrs教授在Nature Chemical Biology【11】发表文章发现,哺乳动物基因组中的6mA含量很低(每百万个A中仅6~13个携带6mA修饰),并且这些6mA并不是由甲基化酶催化的,而是来自m6A RNA的甲基分解代谢和再利用,由DNA聚合酶将携带6mA的核苷酸整合到基因组中。

Nature Chemical Biology同期配了来自英国牛津大学的Paolo spingardi和skirmantas Kriaucionis对该研究的评论“How m6A sneaks into DNA”。他们指出,DNA修饰的低丰度,并不能成为我们质疑其生物学功能的论据,但是当某种修饰的含量非常稀有时(例如6mA在哺乳动物基因组中的含量),就可能使其在细胞中的功能受到挑战

并且,6mA通过核酸代谢回收途径被整合到基因组中,使得它难以成为一种表观遗传标记。对于在哺乳动物中具有重要功能的DNA 5mC修饰,细胞有一套机制严格地限制来自核酸分解代谢产生的5mC掺入基因组中,以确保其精确的定位。而6mA掺入基因组似乎没有被限制,这提示6mA本身的分布(在基因组上的精确定位),可能并不重要。

4月,中国科学院的汪海林教授(2015年与陈大华教授合作在Cell发表果蝇中DNA 6mA鉴定研究)在Cell Research也发文报道6mA通过DNA聚合酶X家族成员非模板依赖的Polyλ掺入基因组中【12】。在体外实验中,研究者将N6mdATP代替部分dATP与高保真DNA聚合酶Taq(模板依赖的聚合酶)共同孵育,发现6mA可被掺入PCR反应中;而当N6mdATP与dATP的比例降低至1:1000时,PCR产物中则检测不到6mA,这说明N6mdATP并不是一种友好的DNA合成底物,且高保真Taq酶更倾向于使用dATP而非N6mdATP

而在细胞周期的G1期,非模板依赖的Polyλ将6mA核苷掺入基因组;敲低Polyλ可显著降低G1期和sub G1期6mA的水平。同时,研究者利用CRISPR敲除潜在的6mA甲基转移酶METTL4和去甲基酶ALKBH1后(关于催化DNA 6mA的酶目前并没有定论,还处于争议之中),发现UPHLC-MS/MS分析方法并不支持它们是6mA相关催化酶,因为6mA的水平均未发生明显变化(详见BioArt报道:

)。值得注意的是,该研究未发现DNA 6mA可通过RNA m6A代谢掺入,与前述Nature Chemical Biology论文中的结论相左。因此,关于基因组中低丰度的6mA来源,还需进一步的研究。

四、柳暗花明?6mA作为表观遗传标记参与线粒体生命活动的调节

目前6mA的研究不仅仅是它本身的鉴定和功能研究,更涉及到了6mA甲基化酶和去甲基化酶的鉴定,并且敲低或过表达这两类酶,也成为研究6mA功能的重要手段。如果6mA在基因组中的含量很低,那么这些酶在细胞中的真实底物是什么呢?

事实上,关于6mA的甲基化酶和去甲基化酶,学界也颇多争议。

2016年,Andrew Z. Xiao教授在小鼠中鉴定ALKBH1为6mA去甲基化酶【6】。2018年,晏光荣肖传乐教授则鉴定了N6AMT1作为人的6mA甲基化酶【7】。而谢琦教授等以神经胶质瘤为模型,发现N6AMT1并不具有6mA甲基化酶活性,而ALKBH1具有去甲基化酶活性(该研究为谢琦,Jeremy N. rich和Andrew Z. Xiao两方课题组合作完成)【8】

2019年,南加州大学医学院的Douglas E. Feldman教授将METTL4和ALKBH4分别鉴定为6mA修饰的甲基化酶和去甲基化酶(但是并没有提及N6AMT1和ALKBH1)(详见BioArt报道:

)。

今年2月,清华大学的李海涛教授和Andrew Z. Xiao教授,中国农业大学的陈忠周教授与中科院生物物理所闫小雪分别在Cell Research发表研究,解析了ALKBH1作为哺乳动物DNA 6mA去甲基化酶的自由态以及与DNA底物结合后的复合物结构及工作机制(详见BioArt报道:

Cell Research背靠背 | 李海涛、陈忠周团队分别发文揭示哺乳动物中DNA 6mA去甲基化酶ALKBH1的工作机制​mp.weixin.qq.com

)。

蛋白结构是功能的基础,结构解析往往为我们了解蛋白提供了“眼见为实”的证据。由此,虽然6mA甲基化酶尚存争议,但似乎ALKBH1是6mA去甲基化酶,至少在体外实验中证明ALKBH1是6mA去甲基化酶。而6mA在哺乳动物基因组中的含量非常稀少,ALKBH1作为去甲基化酶,能发挥多大的生物学功能呢?一时间,疑问更多了。

今年3月,芝加哥大学的何川教授在Molecular Cell发表的研究为6mA研究提供了新的方向【15】(详见BioArt报道:

)。该研究发现哺乳动物中的6mA修饰主要集中在线粒体DNA上(平均每个mtDNA分子有4个6mA),而核基因组DNA的6mA量极低。同时,研究者鉴定了主要定位在线粒体中的METTL4,是6mA的甲基转移酶(这与南加州大学医学院的Douglas E. Feldman教授的结论一致,但他们当时认为METTL4介导核基因组的6mA修饰。

但是该结果与最近施扬教授团队发表在Cell Research上的结果相左,施扬团队证明METTL4催化的底物是RNA而不是DNA)。6mA阻碍线粒体转录因子结合mtDNA,从而减弱mtDNA的转录和拷贝数(mtDNA copy number)。在低氧情况下,6mA的水平上升,提示6mA与线粒体压力应答过程相关。

那么,ALKBH1是否定位在线粒体中并发挥mtDNA 6mA去甲基化酶的功能呢?已有研究表明ALKBH1可以定位在线粒体中,功能包括调控线粒体tRNAMet的2’-O-甲基化【15】,线粒体RNA granule的形成【16】和拷贝数【17】等。而关于ALKBH1是否调控mtDNA 6mA尚不清楚。

综上,关于N6AMT1和ALKBH4,ALKBH1的功能,它们真正的催化底物,都需要进一步研究。

五、雄关漫道真如铁,而今迈步从头越

从2015年首次在真核生物中被鉴定到如今5年多的时间里,6mA获得了广泛的关注,也引发了激烈的争议。我们对6mA的态度也从期待变成了审慎。6mA在哺乳动物中的低丰度,与其高度的动态性有关吗?由DNA聚合酶掺入的6mA是随机分布的吗?是否具有生物学功能?目前鉴定的m6A相关催化酶,是真实的吗?

目前的表观遗传研究为我们回答这些问题,提供了一些线索,当然也引发新的疑问。

1)6mA的高度动态性;

在果蝇胚胎中,6mA的丰度在胚胎发育的0.75h内达到顶峰(~0.07%,6mA/dA),而后迅速降低,4h后仅剩~0.001%【3】,另外两篇文章也提到了斑马鱼的猪的胚胎发育过程以及小鼠应对环境变化中DNA 6mA具有高度动态性【20,21】,由此可见,6mA的丰度呈高度动态性,对其鉴定也需要把握时机。

而目前哺乳动物中6mA的鉴定多集中HeLa,HEK293T和mES等有限的细胞系中,这可能导致无法真实反应6mA在哺乳动物中的丰度。因此,为更准确的了解哺乳动物中6mA的含量,还需扩大检测范围(如检测各发育时期中6mA的丰度)。

2)单一位点的单个修饰也有大功能;

虽然有关6mA的丰度尚存争议,但是高精度质谱检测已提示6mA在哺乳动物中的含量非常有限,这也为6mA的生物学功能画了问号,丰度低就说明没有什么大功能吗啊?那么,表观遗传修饰的丰度和生物学功能间是怎样的关系呢?

今年5月,澳大利亚新南威尔士大学的Merlin Crossley 教授在Nature Communication发文“Methylation of a CGATA element inhibits binding and regulation by GATA-1”【18】,该研究发现c-kit基因座位的单个5mC甲基化位点就足以影响转录因子GATA-1对该基因座位的结合,并调控造血系统的正常发育

这从某种程度上说明低丰度的表观遗传修饰也可通过影响某一生物过程的关键基因而发挥巨大作用。因此,为探索6mA的生物学功能,开发可信赖的6mA分布位点检测方法是十分必要的。

六、关键论文作者的回应

6mA研究出现了很多的争议和讨论,Bioart联系上述研究中的多位相关人士了解他们对于该领域的看法,有永远不信的、有将信将疑的、有绝对相信的,当然还有一直观望看戏的。此外,BioArt特别邀请了两位直接参与实验的关键论文第一作者Tao P. Wu博士和Eric L. Greer博士,谈谈他们对6mA的鉴定和相关催化酶的看法。

Tao P. Wu博士(现在贝勒医学院人类与分子遗传系建立实验室)早前在耶鲁大学的Andrew Z. Xiao教授实验室做博后,是6mA在小鼠胚胎干细胞中的鉴定和发现ALKBH1是6mA去甲基化酶的研究的第一作者【6】,同时也是2018年Cell论文(6mA修饰与胶质瘤)的共同第一作者【8】Tao P. Wu博士关于小鼠ESC中6mA含量低和受质谱中核酸酶的污染等问题做出了回应:

“虽然越来越多的证据显示m6dA是哺乳动物中一种功能性的DNA表观修饰,但是仍然有来自阴性结果的质疑,需要进一步探讨。在我们发表第一篇Nature文章之后,德国的Thomas Carell研究小组发文声称在wild-type mouse ES cell 中检测不到m6dA (with UPLC-MS/MS)。读者如果熟悉干细胞研究相关背景的话,应该很清楚小鼠干细胞的培养有多种条件与方法,其中尤其以培养基对细胞状态的影响最大。

Andrew Z. Xiao实验室正在撰文,描述它们对DNA修饰的影响。最近,Eric Greer小组(postdoc in Yang Shi Lab before)又发表结果,声称DNA digestion enzyme含有来自细菌DNA 的污染。这在我们和其他几个实验室做UPLC-MS/MS检测的时候,都没有发现类似的结果;相关的对照实验结果我们也会放在未来的文章当中。

对于SMRT Sequencing, Shijia Zhu & Gang FangGenome Research文章中已经进行过讨论(Nat Rev Genet发表房刚组细菌表观组综述论文),在m6dA丰度极低的情况下,SMRT 会产生假阳性结果。这也正是为什么,该领域亟需新的方法和研究模型。最近,肖传乐 & Kai Wang发表文章,尝试了利用机器学习算法分析Nanopore sequencing数据来读取DNA修饰信息。Tao P. Wu实验室也在开发新的方法,拓展新的研究模型。”

Eric L. Greer博士早前是施扬教授的博士后,鉴定了线虫中的6mA【4】,并发现哺乳动物的6mA可能来源于核酸酶的污染。Eric L. Greer博士目前在哈佛医学院独立开展研究工作,就目前鉴定的6mA相关催化酶的看法如下:(原文附在译文之后)

“就哺乳动物6mA催化蛋白的酶活性而言,这些蛋白都是我们之前在线虫中鉴定的6mA相关催化酶的同源蛋白。我们在体外条件下得到的酶活性不能真实反应生理条件下该酶的催化活性,但是实验间的平行性差(当我们有关线虫中6mA及相关催化蛋白的文章出来时,我们已鉴定了哺乳动物中6mA催化酶,只是这些结果太初步了)。

这可能是由于我们未得到最佳的酶反应条件,也可能这些酶有其他的更主要的作用底物,而当过表达它们到过高的浓度时,它们也可以完成N6位的甲基化和去甲基化过程。如果这些酶最主要的底物是其他物质(比如RNA或者其他DNA修饰),当在某一体系中过表达超高浓度的你感兴趣的蛋白,来迫使它们发挥功能时,你可能被结果所欺骗,认为找到了正确的底物。这就是为什么进行酶活性动力学实验非常重要,这有利于确认它们在生理条件下的活力。”

以下为邮件原文:In terms of the enzymatic activity of the mammalian proteins these have all been identified as homologues of proteins that we identified in C. elegans. We have found that we get spurious activity of these proteins in vitro but it is not consistent from experiment to experiment (we had actually identified the mammalian proteins at the same time when our C. elegans story came out but thought they were too preliminary). This could reflect that the enzymatic conditions are not optimal or that these enzymes have another primary substrate and when forced in excessive concentrations to methylate or demethylate N6 on adenosine they will. If the primary target of the enzyme is something else (say RNA or another DNA modification) you can still force activity by overloading the system with really high concentrations of your enzyme of interest and trick yourself into thinking that it is the right target. That is why it is important for showing methylases and demethylases to perform kinetic experiments to determine whether the enzymatic activity would be physiologically relevant.)

实际上,不仅是6mA相关催化酶,包括目前最受关注的RNA m6A修饰,其去甲基化酶FTO的身份也经历了多次质疑和反转(详见BioArt报道:

NCB | 这一次,FTO是snRNA的m6Am去甲基化酶 )。由此可以看出,甲基化酶和去甲基化酶可能同时存在多种催化底物,比如FTO可同时介导m6A和m6Am去甲基化,解析其生理条件下最主要的催化底物才是关键。

此外,BioArt编辑部也注意到在国家自然科学基金中,有235万元的项目涉及6mA,包括6mA在植物和动物中的多项研究。鉴于6mA的争议和其在线粒体稳态维持的功能,今后的研究需秉持更加严谨和科学的态度,为我们揭开高等生物中6mA的全貌。

编后记:对于争议领域来说,正常的学术讨论是非常有必要的,讲究“有一份证据说一分话”,最好做到不打棍子,不扣帽子。新兴领域出现争议是再所难免的,面临争议就需要更多的研究同行携手探求科学的本质,努力就本领域的问题达成共识,良性的学术讨论环境才能促进领域扎实的向前发展。

BioArt特别提醒有兴趣研究DNA 6mA生物学功能的实验室,从事DNA 6mA有较高的门槛,目前除了高灵敏度的质谱技术(排除了可能污染)以外,基于抗体、三代测序的技术都存在很大的不确定性,再者围绕6mA修饰的相关酶类也存在极大的争议,因此,围绕6mA开展生物学功能研究的时候都需要采取审慎的态度,切勿盲目跟风下结论。

围绕6mA修饰的讨论还会持续下去,就这个话题BioArt也非常欢迎相关专家来稿发表不同的观点和意见。上述文章也并未对每一篇相关文章都进行了展开,限于学识和水平,文中错误疏漏之处在所难免,还请读者包容!

编者特别感谢一年多来,何川(芝加哥大学)、施扬(哈佛医学院)、陈大华(中科院动物所)、李海涛(清华大学)、伊成器(北京大学)、杨运桂(中科院基因组所)、汪海林(中科院生态环境研究中心)、房刚(美国西奈山医学院)、蓝斐(复旦大学)、吴涛(贝勒医学院)、谢琦(西湖大学)、刘颖(北京大学)、骆观正(中山大学)、陆发隆(中科院遗传发育所)、沈立(浙江大学)、Eric L. Greer(哈佛医学院)等多位专家就DNA 6mA修饰相关研究坦诚地提供相关信息和个人观点,部分专家审阅全文并提出了宝贵意见。

制版人:玉壶

参考文献

[1]D. J. Weisenberger, M. Campan, T. I. Long, M. Kim, C. Woods, E. Fiala, M. Ehrlich,P. W. Laird, Analysis of repetitive element DNA methylation by MethyLight. Nucleic Acids Res.33, 6823–6836 (2005)

[2] Fu, Y., Luo, G.Z., Chen, K., Deng, X., Yu, M., Han, D., Hao, Z., Liu, J., Lu, X.,Dore, L.C., et al. (2015). N6-methyldeoxyadenosine marks active transcription start sites in Chlamydomonas. Cell 161, 879–892.

[3] Zhang, G., Huang, H., Liu, D., Cheng, Y., Liu, X., Zhang, W., Yin, R., Zhang, D.,Zhang, P., Liu, J., et al. (2015). N6-methyladenine DNA modification in Drosophila. Cell 161, 893–906.

[4] Greer, E.L., Blanco, M.A., Gu, L., Sendinc, E., Liu, J., Aristiza´ bal-Corrales, D., Hsu, C.H., Aravind, L., He, C., and Shi, Y. (2015). DNA methylation on N6- adenine in C. elegans. Cell 161, 868–878.

[5] Holger Heyn, Manel Esteller, An Adenine Code for DNA: A Second Life for N6-Methyladenine, Cell. 2015 May 7;161(4):710-3.

[6]T. P. Wu, T. Wang, M. G. Seetin, Y. Lai, S. Zhu, K. Lin, Y. Liu, S. D. Byrum, S. G. Mackintosh, M. Zhong, A. Tackett, G. Wang, L. S. Hon, G. Fang, J. A. Swenberg, A. Z. Xiao, DNA methylation on N6-adenine in mammalian embryonic stem cells. Nature 532, 329–333 (2016)

[7] C. L. Xiao, S. Zhu, M. Heet al. N(6)-Methyladenine DNA Modification in the Human Genome. Mol Cell,2018,71(2):306-318

[8] Xie, Q., Wu, T.P., Gimple, R.C., Li, Z., Prager, B.C., Wu, Q., Yu, Y., Wang, P.,Wang, Y., Gorkin, D.U., et al. (2018). N6-methyladenine DNA modification in glioblastoma. Cell 175, 1228–1243.

[9] Zhou C, Wang C, Liu H, Zhou Q, Liu Q, Guo Y, Peng T, Song J, Zhang J, Chen L, Zhao Y, Zeng Z, Zhou DX, Identification and analysis of adenine N6-methylation sites in the rice genome. Nat Plants. 2018 Aug;4(8):554-563.

[10]QianZhang,ZheLiang,XueanCui,ChangmianJi,YunLi,PingxianZhang,JingrongLiu,AdeelRiaz1PuYao,MinLiu,YunpengWang,TiegangLu,HaoYu,DongleiYang,HongkunZheng,XiaofengGu,N6-Methyladenine DNA Methylation in Japonica and Indica Rice Genomes and Its Association with Gene Expression, Plant Development, and Stress Response, Molecular Plant, Volume 11, Issue 12, 3 December 2018, Pages 1492-1508.

[11] Z. Liang, L. Shen, X. Cui, S. Bao, Y. Geng, G. Yu, F. Liang, S. Xie, T. Lu, X. Gu, et al.DNA N6-adenine methylation in Arabidopsis thalianaDev. Cell, 45 (2018), pp. 406-416

[12] Liu X, Lai W, Li Y, Chen S, Liu B, Zhang N, Mo J, Lyu C, Zheng J, Du Y R, Jiang G, Xu G L, Wang H. N6-methyladenine is incorporated into mammalian genome by DNA polymerase. Cell Res., 2020 Apr 30. doi: 10.1038/s41422-020-0317-6.

[13] M. J. Koziol, C. R. Bradshaw, G. E. Allen, A. S. H. Costa, C. Frezza, J. B. Gurdon, Identification of methylated deoxyadenosines in vertebrates reveals diversity in DNA modifications. Nat. Struct. Mol. Biol. 23, 24–30 (2016)

[14] Alexandre Hofer, Zheng J. Liu, and Shankar Balasubramanian, Detection, Structure and Function of Modified DNA Bases, J. Am. Chem. Soc. 2019, 141, 6420−6429

[15] Kawarada L, Suzuki T, Ohira T, Hirata S, Miyauchi K, Suzuki T, ALKBH1 is an RNA dioxygenase responsible for cytoplasmic and mitochondrial tRNA modifications. Nucleic Acids Res. 2017 Jul 7;45(12):7401-7415.

[16] Wagner A, Hofmeister O, Rolland SG, Maiser A, Aasumets K, Schmitt S, Schorpp K, Feuchtinger A, Hadian K, Schneider S, Zischka H, Leonhardt H, Conradt B, Gerhold JM, Wolf A, Mitochondrial Alkbh1 localizes to mtRNA granules and its knockdown induces the mitochondrial UPR in humans and C. elegans. J Cell Sci. 2019 Oct 1;132(19).

[17] Müller TA, Struble SL, Meek K, Hausinger RP. Characterization of human AlkB homolog 1 produced in mammalian cells and demonstration of mitochondrial dysfunction in ALKBH1-deficient cells. Biochem Biophys Res Commun. 2018 Jan 1;495(1):98-103

[18].Yang, L., Chen,Z., Stout, E.S. et al. Methylation of a CGATA element inhibits binding andregulation by GATA-1. Nat Commun 11, 2560 (2020).https://doi.org/10.1038/s41467-020-16388-1

[19].Ma, C., Niu, R., Huang, T., Shao, L. W., Peng, Y., Ding, W., … & Liu, Y. (2019). N6-methyldeoxyadenine is a transgenerational epigenetic signal for mitochondrial stress adaptation. Nature cell biology, 21(3), 319.[20].Liu, J., Zhu, Y., Luo, G. Z., Wang, X., Yue, Y., Wang, X., … & He, C. (2016). Abundant DNA 6mA methylation during early embryogenesis of zebrafish and pig.Nature communications, 7(1), 1-7.[21].Yao, B., Cheng, Y., Wang, Z., Li, Y., Chen, L., Huang, L., … & Jin, P. (2017). DNA N6-methyladenine is dynamically regulated in the mouse brain following environmental stress.Nature communications, 8(1), 1-10.

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Addgene: dCAS9-VP64_GFP

dCAS9-VP64_GFP (Plasmid #61422)

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Plasmid 61422

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Concentrated Lentiviral Prep 61422-LVC

Virus (50µL at titer ≥ 2.5×10⁶ TU/mL)

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  • dCAS9(D10A,H840A)-VP64_2A_GFP

  • Synthetic; S. pyogenes

  • D10A and H840A in Cas9

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Ready-to-use Concentrated Lentiviral Prep particles produced from dCAS9-VP64_GFP (#61422). In addition to the viral particles, you will also receive purified dCAS9-VP64_GFP plasmid DNA.

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  • For your References section:

    Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex.
    Konermann S, Brigham MD, Trevino AE, Joung J, Abudayyeh OO, Barcena C, Hsu PD, Habib N, Gootenberg JS, Nishimasu H, Nureki O, Zhang F.
    Nature. 2014 Dec 10. doi: 10.1038/nature14136.
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    PubMed 25494202

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泰热I代 TALE-VP64人工转录激活因子–用于基因过表达–特别适合大基因(>3k)研究

泰热I代 TALE-VP64人工转录激活因子–用于基因过表达–特别适合大基因(>3k)研究

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100ug

        可用于任意目的基因的过表达,特别是3k以上的大基因。VP64是一个转录激活结构域,TALE-VP64结合到目的基因启动子序列上以后可以增强目的基因的转录水平来增加目的基因表达。用TALE-VP64来进行过表达的优点:         1:可用于细胞内任意基因的过表达,所过表达的对象是细胞内源性的基因,没有其它的修饰和副产物的出现;         2:不受克隆和模板的限制,周期短,而有一些基因比如大基因,高GC含量基因,它们的克隆往往是很困难的;         3:不同的过表达TALE-VP64载体的表达增强效果会有差异,可以用来做表达强度效应的实验,得到目的基因激活程度不同工具或细胞群体。也可以通过结合不同TALE-VP64分子协同作用提高转录水平。A,B,C,D分别代表设计的4种用于过表达的TALE-VP64人工转录因子,A+D为同时转染两者,在293细胞中同时转染4种质粒可以激活基因表达10000多倍。Perez-Pinera P et al. Synergistic and tunable human gene activation by combinations of synthetic transcription factors. Nat Methods. 2013 Mar;10(3):239-42.用TALE人工转录因子过表达miRNA,紫色代表转录激活域为P65,绿色部分为VP64,miR1代表转染用于过表达的1号质粒,依次类推。将所有质粒同时共转染可以增强miRNA过表达250倍以上。Maeder ML et al. Robust, synergistic regulation of human gene expression using TALE activators. Nat Methods. 2013 Mar;10(3):243-5.服务内容:针对目的基因设计并构建一套4个TALE-VP64载体,提供给客户50ug质粒实物及测序结果和报告。载体选择:我们提供带有不同荧光表达的载体用于构建,另有带有抗性载体可以进行筛选。

规格
100ug

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