分子生物学 | 常见实验技术 | Plasmids | Cloning Vector | Reporter

 

2023年02月03日

换了个实验室，我还是唯一的搞生信的，但明显学习氛围非常浓厚，有实验的问题也基本能够得到解答，非常开心，开始深度学习实验技能。

先从质粒开始，plasmid。

Plasmids | Cloning vectors: Plasmids | Why do we use plasmids in RDT? | features of a plasmid

质粒是最常见且有效的DNA载体，可以承载GOI，也可以是工具类DNA，如gRNA，里面最少包含启动子、限制性内切酶位点，GOI，选择marker，抗性基因。

OK，然后再从这篇文章来看一些技术：OCA-T1 and OCA-T2 are coactivators of POU2F3 in the tuft cell lineage【一个经典的coactivator的研究】

基于plasmid的技术：

Luciferase reporter assay | What is luciferase assay used for? | Applications of Luciferase assay

• 很简单的系统，一个promoter+一个荧光素酶（readout），可以做很多事情，比如promoter活性强弱，比如TF+coactivator的活性。

Protein tagging | What is the purpose of tagging proteins? | Applications of protein tagging

• 只需要在plasmid的ORF后面加一个tag就可以做很多事情，比如标记特定蛋白，后面可以接WB等等。
• What is an epitope tag? 常见的tag有HA、V5、Biotin、His
• Design Primers with a His-tag for YGOI and Insert to Plasmid

Techniques to study DNA protein interaction 

• 研究DNA和蛋白质互作的方法，比如TF
• GST pulldown | GST Pull Down Assay | Protein-Protein Interaction Using Pull Down Assay 【在细菌里研究两个蛋白的互作，GST tag pulldown，+ -一比就知】

Gel filtration chromatography in 5 minutes | Size Exclusion Chromatography | Lab Procedure【凝胶电泳类】

sds-page是sds聚丙烯酰胺电泳实验，单纯只是电泳。WB指通过抗体特异性杂交目的蛋白质显影的实验。一般作wb之前需要做sds-page把蛋白按质量大小跑开，之后转膜杂抗体。但sds- page之后可以不接wb，直接把胶染考马斯亮蓝看蛋白质条带（非特异性）。

SDS-PAGE是一种基于不同分子量分离蛋白的实验方法。Western Blot是一种从蛋白混合物中半定量分析某种特定目标蛋白的分析技术。WB的第一步通常用SDS-PAGE来分离蛋白。但后续还包括将胶里的蛋白转到NC膜或PVDF膜上，封闭，一抗二抗封闭，显影这些步骤。

小知识：抗体通常是识别多肽polypeptide的【5个AA，二维】，而不是三维domain，这就是为什么可以接着SDS-PAGE做抗体检测。

SDS-PAGE explained - Protein Separation Technique

Immunoprecipitation (IP) principles and troubleshooting【免疫沉淀，研究蛋白互作利器】

input：没做免疫共沉淀的总的蛋白，whole cell lysate

input是阳性对照的意思。免疫共沉淀实验是对蛋白A进行IP，对蛋白B进行WB。而Input是直接取细胞裂解液，进行蛋白B的WB，证明细胞裂解液中存在WB。

重点：生物学实验里总是会设置positive control和negative control，以增加目标信号的可靠性。

DNA pull-down assays

• Pull-down assays are used to selectively extract a protein–DNA complex from a sample. Typically, the pull-down assay uses a DNA probe labeled with a high affinity tag, such as biotin, which allows the probe to be recovered or immobilized. A biotinylated DNA probe can be complexed with a protein from a cell lysate in a reaction similar to that used in the EMSA and then used to purify the complex using agarose or magnetic beads. The proteins are then eluted from the DNA and detected by western blot or identified by mass spectrometry. Alternatively, the protein may be labeled with an affinity tag, or the DNA–protein complex may be isolated using an antibody against the protein of interest (similar to a supershift assay). In this case, the unknown DNA sequence bound by the protein is detected by Southern blotting or through PCR analysis.

MicroScale Thermophoresis (MST Service) 

• a biophysical technique that measures the strength of interaction (affinity) between two molecules by detecting variations in fluorescence signal as a result of an IR-laser induced temperature change.

 

 

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搞生物其实门槛非常高，基因分子都是看不见摸不着的，生物技术是唯一的观测方法，这也就是生信为什么这么依赖生物，不做实验你连材料都没有，常规的裸数据挖掘都是二流的生信。

做dry lab的读文章时看到各种实验技术老是一头雾水，非常有必要科普下那些经常出现在paper里的实验技术。dry lab要想做得好，必须要精通实验技术，了解他们的核心需求！！！

如果你只能做一些常规的生信分析，那你注定要被web lab的嫌弃；如果你的生信分析能搞出一些fancy和novel的东西，那你就可以牵着web lab的鼻子走了，生信分析的power是非常强大的，有些甚至已经达到dominate的程度了。

TOC

• Reporter genes、Reporter Lines、reporter mouse models 报告基因
• Transgenic Mouse model 转基因小鼠模型及其基因型书写代号：Tg(GBS-GFP), Sufu^f/f, Gli3^D699, Ptch1^f/f, b3-IIIa-Cre, and Wnt1-Cre
• Cell lines、primary cell 细胞系、原生细胞
• Cre/lox system

 

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FACS

flow cytometry

FACS

Flow Cytometry & FACS | Beginner Data Interpretation Tutorial - YT

flow cytometry翻译就是流式细胞术，用来做细胞计数的

FACS在此基础之上，用电极来收集特定两个维度的细胞，衡量细胞特征的参数很多，设计抗体则可以做任何细胞表面蛋白的筛选。

 

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immunohistochemical analysis (IHC)

Immunohistochemistry | How to perform immunohistochemistry? | application of immunohistochemistry - YT

primary antibody

secondary antibody

最大的难点是要了解组织切片的细胞结构，否则就是看个热闹，对于结构复杂的组织（比如心脏），看图将会非常难。

To validate these observations, we performed immunohistochemical analysis of embryonic guts at E11.5–E14.5 with GFP and various differentiation markers. At E11.5, GFP signals were detected in the progenitor populations, mainly in mBP (Sox10+Phox2b+Tuj1–) (Figure 2F, filled arrowheads), whereas low levels of GFP could also be observed in some mGP (Sox10+Phox2b–) (Figure 2F, open arrowheads). Nevertheless, GFP signal was not found in the fully differentiated cells expressing high levels of Tubb3 (Tubb3high) or other late-stage neuronal (Elval4) and glial markers (Fabp7) in E14.5 guts (data not shown).

这里就用了连个antibody（Sox10+Phox2b+），两种颜色，推测出三种细胞类型，BP、NP、GP，荧光GFP标记的是Hh激活的细胞，最后根据这些信息做出推理，有时也会对细胞数量和荧光density做出柱状图量化。

Next, we examined how alterations of hedgehog pathway activity affect the cell-state transition of enteric NC in mice. The hedgehog pathway was activated in mice by the deletion of Ptch1 (bIII3a-Cre;Ptch1f/f) or Sufu (Wnt1-Cre;Sufuf/f) as well as suppressed by the constitutive expression of a truncated GLI3 repressor in Gli3Δ699/Δ699 mice. As these mutants died on or before E13, as described previously,5,8 E12.5 guts were collected and the BP, neuronal progenitor, and GP were identified based on the expression of Sox10, Phox2b, and/or Tubb3 (Tuj1). In addition to an early emergence of the mGP (Figure 2G, open arrowheads) in the Ptch1 and Sufu mutant guts as described previously,5,8 we found that the mNPlate (Figure 2G, dark pink arrowheads) neuronal progenitor populations are increased with concomitant reductions of the mNPearly (Figure 2G, light pink arrowheads) and mBP (Figure 2G, white arrows) cells in both Ptch1 and Sufu mutants (Figure 2G). Conversely, constitutive suppression of the hedgehog pathway significantly increased the mBP population and reduced the number of mGP cells in Gli3Δ699/Δ699 mice (Figure 2G). Taken together, these data suggest that hedgehog pathway activation promotes the progression of bipotent enteric NCs to differentiate into more mature cell states. 

 

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Reporter genes、Reporter Lines、reporter mouse models

Reporter (gene)，报告基因，a gene that researchers attach to a regulatory sequence of another gene of interest. the characteristics they confer on organisms expressing them are easily identified and measured, or because they are selectable markers.

特征：1.容易观测得到，比如荧光蛋白基因green fluorescent protein (GFP)【which causes cells that express it to glow green under blue light】；2.在被插入生物里不存在。

使用方法：1.单纯的当一个marker，通过插入到指定的调控原件（promoter）后，可以得知该基因的表达情况；2.如果reporter后面带一个基因，那么就可以观察这次转基因是否成功。

核心：reporter因为其容易被观察测量的特性，可以灵活准确地分析我们的转基因过程。

举例1：Many methods of transfection and transformation – two ways of expressing a foreign or modified gene in an organism – are effective in only a small percentage of a population subjected to the techniques.[12][13] Thus, a method for identifying those few successful gene uptake events is necessary. Reporter genes used in this way are normally expressed under their own promoter (DNA regions that initiates gene transcription) independent from that of the introduced gene of interest。转基因的效率不是100%的，有时甚至很低，所以可以用reporter来筛选。

举例2：Reporter genes can be used to assay for the expression of a gene of interest that is normally difficult to quantitatively assay. 一些特别难做测序的基因可以直接用GFP看表达。

举例3：Reporter gene assay have been increasingly used in high throughput screening (HTS) to identify small molecule inhibitors and activators of protein targets and pathways for drug discovery and chemical biology.

参考：Reporter gene - wiki 

Overview of the reporter genes and reporter mouse models 

Generation of Transgenic Mouse Fluorescent Reporter Lines for Studying Hematopoietic Development

Gene Regulatory Logic for Reading the Sonic Hedgehog Signaling Gradient in the Vertebrate Neural Tube

 

Transgenic Mouse model

不搞生物的看到这些代号的时候总是一脸雾水，尼玛，搞生物的怎么比搞数学的符号还要复杂。其实一点都不复杂，只是需要掌握它们的规则。

问题描述：rules and nuances of strain and gene nomenclature，how genotypes of individual animals should be designated/symbolized

 

 

 

参考：Mouse Genetic Nomenclature: Standardization of Strain, Gene, and Protein Symbols

DESIGNATING GENOTYPES: WHAT DOES '+' REALLY MEAN?

CRE LOX BREEDING FOR BEGINNERS, PART 1

 

 

 

 

 

Wnt1-Cre transgenic mouse model

Cre-LoxP Recombination

lineage tracing by Cre-LoxP system - A step by step discussion

什么是Cre？

recombinase enzyme isolated from bacteriophage P1 which targets LoxP DNA sequences for the process of recombination

In the Cre mouse every cell contains the coding sequence of Cre, but only cells expression protein X will express the Cre

In the reporter mouse every cell contains the coding sequence of GFP, but none of them give GFP signal because of presence of STOP sequence.

heterozygous mouse

Once the stop sequence is removed from stem cell DNA then every cell originated from stem cell will give positive GFP signal.

从而达到lineage tracing的效果。

 

Wnt1^Cre/R26R^Tomato mouse embryos - 为什么用这个老鼠？

ROSA26 is a locus used for constitutive, ubiquitous gene expression in mice. It was first isolated in 1991 in a gene-trap mutagenesis screen of embryonic stem cells (ESCs). Over 130 knock-in lines have been created based on the ROSA26 locus.

Wnt1 - 为什么用这个基因？

Wnt1^Cre recombines in the dorsal neural tube where the premigratory neural crest resides.

用这个技术标定了这个细胞家系（premigratory neural crest），然后用单细胞技术measured mRNAs of single TOMATO⁺ cells

 

asynchrony - 异步的：与同步相反，不在同一时间发生。

anteroposterior axis of the embryology，通俗点就是从头到尾。

dorsal to ventral，从背到腹部。

epithelial-to-mesenchymal transition (EMT) 上皮到间叶细胞的转换。

 

 

 

 

Immunohistochemistry - 发育生物学的主要利器，拍片。

Immunohistochemistry - YouTube

根据感兴趣的分子（即抗原，antigen），找对应抗体，一级抗体负责找到我们的antigen，然后加入二级抗体（包含了一个结合抗体和一个催化酶，如HRP酶），最终加入DAB，DAB被局部催化反应形成带颜色的复合物，从而被显微镜观察到。

In the schematic illustration (Figure 1) a formalin-fixed paraffin embedded tissue section is stained using a primary antibody directed towards a specific protein target. A solution containing the primary antibody is added to the tissue section and the antibodies are allowed some time to find and bind to their target. After this step, unbound and surplus antibodies are washed away and the secondary antibody is added. The secondary antibody, which carries a linker molecule with horseradish peroxidase (HRP) enzymes, is also allowed some time to bind to the primary antibody, followed by another washing step. After this, 3,3' Diaminobenzidine (DAB) is added. The HRP enzyme transforms the DAB substrate into a brownish precipitate that is deposited in the tissue at the site of the reaction, thus producing a visual representation of where the primary antibody first bound its target.

注：epitopes即是蛋白上的某个部分，相对于抗原。

看一个案例：

A是一个几毫米的图，里面只有两种颜色，红色就是转基因小鼠的颜色，绿色就是SOX10蛋白的颜色；这张图看着立体，是因为扫描了很多层然后通过计算机合成的。SOX10⁺/Wnt1^TOM+标记的就是neural crest cells (NCCs)

B是A里面的切片部分的截面图，大概有三种颜色，红色就是转基因小鼠的颜色，绿色就是SOX10蛋白的颜色，黄色就是都表达的部分。展现了cranial and trunk portions的分离。

目的：表面一个核心的生物学过程，NCCs在头部和躯干里迁移，

 

荧光蛋白的选择：

GFP：绿色的优先级最高，灵敏、剂量需求小

Tomato：红色

YFP：黄色

具体操作时，每一个蛋白都有自己独特的光谱，可以自由的设定颜色，但是通常都会选择红、绿、蓝。

 

7 fluorescent labels：

DAPI：标记细胞核

 

In situ sequencing - 原位测序

spatial single cell sequencing，单细胞级别的测序，通过barcode杂交，得到扩增的指定基因的序列，然后再用荧光去杂交，最终可以在显微镜下看到每一个细胞的基因表达。（指定RNA扩增 和 荧光显微 技术的完美结合）

 

 

mouse lineage

Immunohistochemistry of control Neurog2^CreERT2/+;R26R^YFP/+ (carrying one copy of CreERT2) and mutant Neurog2^{CreERT2/CreERT2};R26R^YFP/+ (carrying two copies of CreERT2) tamoxifen-injected at E9.5 and analyzed at E15.5

上标表示Neurog2基因被CreERT2给替换了，两个上标都是CreERT2则表示Neurog2的两个allele都被替换了，也就是给knock out了。

+：wild type

-：knock out

YFP：插入或替换了YFP

 

一个案例：

 

 

 

 

参考：

图像识别 | AI在医学上的应用 | 深度学习 | 迁移学习

Inducible Cre Mice