对于韩春雨NBT文章无法重复的问题
需要从Figure 1 开始一步步看
作者的contributions
1. C.H. conceived the study and designed the experiments.
文章 “Story” “Idea” 的设计与发起人
2. X.Z.S. provided intellectual advice on the project and experimental
design.
实验设计, “One guide-faithful” -Figure 2d?
3. C.H. performed mammalian genome editing.
(Figure 4,5)
文章里让科研界最感兴趣的结果,也是无法被重复验证的主要问题
4. F.G. performed the BLAST search and the in vitro cleavage experiments.
(Figure 1, supplementary figure 2)
大肠杆菌E coli 表达的蛋白
NgAgo 基因编辑功能的初步 in vitro 数据支持
注:文章中缺乏蛋白表达纯化的数据
5. F.G., F.J. and Y.W. designed and constructed the clones under the
supervision of C.H.
在韩春雨指导下,学生们设计并做的constructs
注:文章中所有的constructs 看来都是学生们在韩春雨指导下做出来的
6. F.J. and Y.W. performed the in vivo cleavage experiments.
(Figure 3)
哺乳细胞表达的蛋白
NgAgo 基因编辑功能的初步 in vitro 及 in vivo 数据支持
注:文章中缺乏蛋白表达纯化的数据
注:文章中缺乏蛋白细胞内定位的数据以及实验详细步骤
7. X.Z.S. and C.H. wrote the manuscript.
“Story”的编写者们
文章图片分析:
1. Figure 1 E coli GST-NgAgo,in vitro assay
- E coli 表达纯化的是 GST-NgAgo, NBT正文以及supplementary中,存在有关于蛋白
表达的数据吗? - 没有找到
- Figure 1b:
E coli expressed NgAgo with either FW or RV guide DNA: nicked target
plasmid (Lanes 5,6)
E coli expressed NgAgo with both FW and RV guide DNA: linearized target
plasmid (Lane 7)
- 支持Figure 1 的数据有 supplementary Figure 2
E coli NgAgo (assuming GST-NgAgo) 55C preloaded with guide
(没有注明是FW or RV guide), 只能nick plasmid
2. Figure 2 Mammalian, FLAG-NgAgo-HA?
- HEK293T 表达纯化的是 FLAG-NgAgo-HA, NBT正文以及supplementary中,存在有关于
蛋白表达的数据吗? - 没有找到
- 这张图是“one guide faithful”原理的数据来源
- Figure 2a ssDNA很泛泛,没有注明是否是 FW+RV
- Figure 2b 注明了是FW guide
3. Figure 3 Mammalian FLAG-NgAgo-HA
- Figure 3a:
HEK293T expressed NgAgo preloaded with FW guide alone: linearized plasmid
(Lanes 1,2,3)
这和Figure 1b 矛盾 也和supplementary figure 2 有矛盾
- 支持Figure 3a 的数据有 supplementary Figure 1
也是只要preloaded with FW guide就可以 linearized plasmid
这和Figure 1b 矛盾 也和supplementary figure 2 有矛盾
- HEK293T 表达纯化的是 FLAG-NgAgo-HA, NBT正文以及supplementary中,存在有关于
蛋白表达的数据吗? - 没有找到
4. Supplementary Figure 4
文章中唯一的关于NgAgo 在哺乳细胞中定位的数据
- Hela cell 表达的NLS-NgAgo蛋白localized to nucleus
- 这个NLS-NgAgo 蛋白是如何被检测到的?
- 原文引用
“NgAgo is directed into nucleus by NLS.
The engineered NLS-NgAgo was transfected to Hela cells and it shows that the
expressed NLS-NgAgo was compartmented in the
nucleus (DAPI+) and the cells maintained normal morphology. Scale bar = 100
μm. Representative figure of 20 independent
experiments.”