2021年06月08日
能量代謝
Glycolysis 糖酵解
Fatty acid metabolism 脂肪酸代謝
Pentose phosphate pathway - PPP - 磷酸戊糖途徑
tricarboxylic acid cycle - TCA - 三羧酸循環
一句話總結：
參考：糖酵解TCA
干細胞領域專有名詞
S/L ESCs, conventional ESCs cultured in serum/LIF (SL) 血清和白血病抑制因子
ground-state ESCs【不同paper叫法不同，S/L and 2i ESCs，CSC paper】
naive, primed and ground state pluripotency
Mouse embryonic stem cells (mESCs) are derived from the inner cell mass (ICM) of pre-implantation stage embryos1,2. mESCs derived in leukemia inhibitory factor (LIF) and serum are representatives of the naive state of pluripotency. The combination of LIF + serum (LS) is used as a “conventional” culture condition for mESCs derivation.【在SL里培養的就是naive，也是conventional】
Until 2008, the derivation of germ-line competent mESCs from non-permissive mice was not possible. However, using the combination of two small molecule inhibitors PD0325901 (PD) and Chir99021 (CH), referred to as “2i”, ESCs could also be derived from non-permissive mouse strains at a higher efficiency5,6. PD and CH are inhibitors of the fibroblast growth factor (FGF) and the glycogen synthase kinase 3 beta (GSK3β) signaling pathways respectively. In mice, pluripotent stem cells can also be derived from the epiblast of post-implantation stage embryos, commonly referred to as epiblast stem cells (EpiSCs). These pluripotent stem cells display primed characteristics and are dependent upon activation of FGF and Activin signaling for their self-renewal7,8. Thus, three distinct pluripotent states, namely naive, primed and ground pluripotency states have been defined in mouse.【用2個抑制劑培養的就是ground state，primed就是用的比較遲的細胞】
參考：
The many faces of Pluripotency: in vitro adaptations of a continuum of in vivo states
Ex vivo (Latin: "out of the living") literally means that which takes place outside an organism. In science, ex vivo refers to experimentation or measurements done in or on tissue from an organism in an external environment with minimal alteration of natural conditions.
Ex vivo means that something is experimented on or investigated outside its natural in vivo environment while in vitro means in the test tube.
2021年05月31日
GO分析后，會出現很多的term，以negative regulation開頭，這是什么意思呢？
negative regulation of biological process
Any process that stops, prevents, or reduces the frequency, rate or extent of a biological process. Biological processes are regulated by many means; examples include the control of gene expression, protein modification or interaction with a protein or substrate molecule.
這表明了方向性，說明這些基因可以抑制某些生物過程。
2021年05月20日
細胞clone
A clone is a group of identical cells that share a common ancestry, meaning they are derived from the same cell.
Broad peaks and Narrow peaks of Histone modifications區別
Different histone mods have very different distributions. For example, "active" marks like H3K4me3 and H3K9ac form sharp peaks at transcription start sites while heterochromatin marks like H3K27me3 can cover blocks of many kb. So which peaks you use for analysis would depend on the modification you're looking at and the questions you're asking. In my experience, the narrow peaks of active marks can be analysed much like the standard analysis of transcription factor binding while you have to be a bit more inventive in analysing marks with broader distributions. I would be very cautious about combining the different peaks though as different distributions may well represent different epigenetic functions.
narrowpeaks can be generally called for TF, since the region bound is pretty much limited. broadpeaks are better for histone modifications or histone modifiers since the regions can be much wider.
TF binding affinity 就是TF結合到DNA序列的一個score 【參考】
massively-parallel reporter assays (MPRA) 大規模平行報告基因分析，利用合成生物學，直接在試管里模擬GWAS，找eQTL。
2021年05月18日
karyotypes 核型，就是染色后直接去看沒有分裂的染色體，是否完整，是否有缺失或三體等。
photographs of chromosomes are taken in order to determine the chromosome complement of an individual, including the number of chromosomes and any abnormalities.
Phased And Unphased Genotypes
phase即分型，在genetic領域特指分出haplotype，雙倍體
Phased data are ordered along one chromosome and so from these data you know the haplotype. Unphased data are simply the genotypes without regard to which one of the pair of chromosomes holds that allele.
A biallelic genotype comes from two chromosomes. Phased means I know not only the genotypes but which chromosome each genotype call came from. This lets you interpret which sets of genotypes are being inherited together
區分：sequencing、genotyping、phasing，先后承接
haplotype frequency：約定N個鹼基，理論上有2的N次方種haplotypes，但實際很少，只有幾種。參考：Statistical Genetics – Part I - CSE Home
GWAS conditional analysis 已經有一個lead SNP，再看其他周邊的SNP在它的條件下是否顯著
I want to test if the SNPs in chr 19 are independent of my top SNP rs2075650.
Conditional analysis to identify the independently associated SNPs, can be done just with the summary statistics using GCTA software.
https://cnsgenomics.com/software/gcta/#Overview
allele-specific expression 很有意思的話題，同一個位點，為什么兩個allele的表達情況不同，顯隱性
Allele-specific expression (ASE) analysis, which quantifies the relative expression of two alleles in a diploid individual, is a powerful tool for identifying cis-regulated gene expression variations that underlie phenotypic differences among individuals.
拓展：Allele-Specific Enhancer Activities，variant也會影響到enhancer的活性
2021年05月12日
干細胞領域
根據全能性的強弱來划分：
- totipotent / omnipotent
- pluripotent
- multipotent
- bipotent
- oligopotent
- unipotent
IPSC - 誘導性多能干細胞【優點：可以從病人的組織重編程得到IPSC，攜帶其全部遺傳物質；缺點：全能性不夠】
Totipotent cells can form all the cell types in a body, plus the extraembryonic, or placental, cells. Embryonic cells within the first couple of cell divisions after fertilization are the only cells that are totipotent. Pluripotent cells can give rise to all of the cell types that make up the body; embryonic stem cells are considered pluripotent. Multipotent cells can develop into more than one cell type, but are more limited than pluripotent cells; adult stem cells and cord blood stem cells are considered multipotent.
EPSC - Expanded Potential Stem Cell - 超潛能干細胞【類似ESC的細胞，這兩個E不是一個意思，不能重編程，只能從受精卵獲取】
We sought to establish cultures of new stem cells from cleavage stage mouse embryos. Under such a culture condition, we speculated that the self renewing stem cell population might have expanded potential as the cells of 4-cell (4C) or 8-cell (8C) embryos or the individual blastomeres retain the potential to differentiate to both the trophoectoderm (TE) and the inner cell mass (ICM)
參考：
基因操作選擇【找不到更好的詞】
Cre-LOX：多用於老鼠模型，可以conditional編輯基因，誘導型。
CRISPR：多用於細胞系，對操作環境和材料要求高，相對簡單的細胞系更容易成功。
2021年04月27日
碰到陌生pathway不要怕，查一下就行了。
membrane potential 膜電勢 - 神經元之間的信號傳導
Neuromuscular junction 肌肉神經接點
Single-cell isolation 單細胞分離技術【認真區分這些技術，不要一團漿糊】
Serial dilution 連續稀釋 - 拿不同的培養液連續稀釋，得到單細胞懸浮液。優點：convenient operation, low cost，in vitro里應用廣泛；缺點：time-consuming，無法精准得到target cells
Micromanipulation 顯微操縱 - 直接在顯微鏡下人工分離，缺點：low throughput
fluorescence-activated cell sorting (FACS) 熒光激活細胞分選術 - 基於flow cytometry【剛科普過】，優點：高通量自動分選；缺點：requires a large number of cell suspensions，不適合分離low-abundance cells，mechanical damage
immunomagnetic separation (IMS) 免疫磁化分離 - magnetic beads磁珠結合到細胞表面的抗體，缺點：操作相對復雜，優點：tumor samples已經應用
laser capture microdissection (LCM) 激光捕獲顯微切割 - 直接在顯微鏡下用激光切割target部位，優點：cancer里廣泛應用；缺點：high cost, low flux, lack of automation, and limited accuracy
microfluidic platforms 微流體平台 - 現在的商業化的標准單細胞分離技術，優點：污染少、試劑便宜；缺點：裝置復雜，門檻較高
參考：Progress and applications of single-cell sequencing techniques
2021年04月20日
Flow cytometry 流式細胞術
用特定的帶熒光的抗體標記細胞，然后用類似單細胞的flow技術來一個一個檢測細胞的信號，一般會選擇兩個維度，最后就畫出了一個二維的散點圖（顏色代表了密度density）。
對於我們的ENCC，則用了 HNK1 和 p75NTR 兩個抗體
The HNK-1 antibody known to define a subpopulation of human lymphocytes with natural killer and killer cell activities was shown to detect a common neuroectodermal antigen. monoclonal antibody HNK-1 is a marker for neural crest cells from the entire rostrocaudal axis.
most caudal neural crest-derived cells co-expressed p75(NTR)-, Phox2b- and Ret-immunoreactivity
用途：
- 從混合細胞中篩選出目標細胞FACS；
- 從相對細胞計數到絕對細胞計數
- 從相對定量到絕對定量分析
- 從單色到多色熒光分析
- 從細胞膜成份到細胞內成份分析
- 液體中可溶性成分的流式細胞分析
- 分子表型分析
參考：
flow cytometry : basic principles - YT - 推薦
immunocytochemistry ICC 免疫細胞化學 - 對象是細胞
immunohistochemistry IHC 免疫組織化學 - 對象是組織
Immunofluorescence IF 免疫熒光 - 泛指
都是用抗體來檢測蛋白的豐度、分布和定位。abundance, distribution, and localization
非常常用的熒光染色劑及其標記物：
- DAPI (blue) - nuclear acid
- SOX10 - dividing ENS progenitor cells
- HUC/D+ immature neurons
- 5-HT+ mature neurons
參考：
ICC vs IHC vs IF – Do You Know the Difference?
2021年04月08日
雙同源重組系統 - 周斌是該領域的大佬
Sox10CreERT2 x R26ReYFP embryos
Wnt1Cre x R26ReYFP embryos
As SOX10 specifically marks dividing ENS progenitor cells, we could retrieve this subpopulation by inducing reporter expression in Sox10CreERT2 x R26ReYFP embryos【這部分是細胞和發育生物學的核心技術手段，門檻極高，必須搞懂。】
Cre-loxP系統有lineage tracing的功能，只要表達過就會一直mark。而直接敲進報告基因則比較局限，只有表達的時候才會mark。
Cre-loxP的幾個核心成分
Cre是recombinase enzymes重組酶，lox是序列位點locus，Cre可以切掉兩個lox之間的DNA序列，這些DNA序列可以很靈活，可以是gene、stop、reporter gene或其他。
Wildly used recombination systems: Cre-loxP, FLP-FRT, Dre-Rox
Cre recombinase is expressed under the control of a tissue- or cell-specific promoter in one mouse line.
Sox10-Cre: Cre gene is knocked into Sox10 locus, after Sox10 promoter, so Cre expression is controlled by Sox10 promoter. Cells with Sox10 expression will also express Cre.reporter gene 報告基因，用法非常靈活，即可以研究特定調控序列的功能，也可以與目標基因耦合，研究基因功能。較常用的報告基因有黃/紅/綠色熒光蛋白（YFP/RFP/GFP）。
Reporter proteins: tdTomato is one of the brightest fluorescent proteins currently available. Other common fluorescent proteins include GFP, EGFP, YFP, EYFP, RFP, CFP etc. as well as non-fluorescent proteins LacZ. The protein product of LacZ gene is β-galactosidase which will react with its substrate X-galactose and generate blue color, can be visualized without fluorescent microscope.
不同的model有不同的用途，主要看marker基因的功能。
The Wnt1-Cre transgenic mouse line is extensively used in the study of the development of the neural crest and its derivatives and the midbrain.
animal model 動物模型，實驗的材料，比如reporter Knockin mouse，fluorescent, bioluminescent proteins or biochemical tags are inserted into the genome. The reporter can replace a gene, be fused to a protein or inserted into the 3' UTR.
參考：
Overview of the reporter genes and reporter mouse models
Dual genetic approaches for deciphering cell fate plasticity in vivo: more than double, 2019 Current Opinion in Cell Biology
Lineage Tracing, primer. 2012 cell - Lineage Tracing through Genetic Recombination
Unravelling cellular relationships during development and regeneration using genetic lineage tracing, 2019 Nature Reviews Molecular Cell Biology
ISH, in situ hybridization 原位雜交
For all ISH analysis, we used Allen Developing Mouse Brain Atlas (http://developingmouse.brain-map.org) and GenePaint (http://www.GenePaint.org).
The Allen Developing Mouse Brain Atlas offers colorimetric In Situ Hybridization data at cellular resolution at seven stages of mouse development.
IHC, immunohistochemistry 免疫組織化學
Immunohistochemistry (IHC) is the most common application of immunostaining. It involves the process of selectively identifying antigens (proteins) in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues.
2021年03月10日
Chromosome banding 染色體條帶
【現在還在用，主要是保證歷史延續性，讓現在的人能看懂古人的研究】
用特定的染色劑處理后的染色體會出現明暗交替的區域，這就是條帶【banding，條帶，名詞】。【染色技術是鼻祖，所以條帶也是一個非常原始的發現，遠遠早於functional annotation】
染色體是細胞內具有遺傳性質的遺傳物質深度壓縮形成的聚合體，易被鹼性染料染成深色，所以叫染色體（由染色質組成）。（英語：chromosome；希臘語：chroma=顏色，希臘語：soma=體）
核小體是染色體結構的最基本單位。核小體的核心是由4對組織蛋白（H2A、H2B、H3和H4）各兩個分子構成的扁球狀8聚體。【與組蛋白修飾密切相關，為什么沒有H1？】在相鄰的連接線之間結合着一個第5種組蛋白（H1）的分子。從染色體的一級結構到四級結構，脫氧核糖核酸分子一共被壓縮了7×6×40×5=8400倍。
1970年后陸續問世的各種顯帶技術對染色體的識別作出了很大貢獻。中期染色體經過DNA變性、胰酶消化或熒光染色等處理，可出現沿縱軸排列的明暗相間的帶紋。按照染色體上特征性的標志可將每一個臂從內到外分為若干區，每個區又可分為若干條帶，每條帶又再分為若干個亞帶，例如“9q34.1”即表示9號染色體長臂第3區第4條帶的第1個亞帶。由於每條染色體帶紋的數目和寬度是相對恆定的，根據帶型的不同可識別每條染色體及其片段。
條帶出現的本質原因？
locus 基因座
一個典型的基因座可能寫成“6p21.3”，解釋如下
6 = 6號染色體
p = 短臂
21 = 2區, 1帶
3 = 子帶3
染色體上的短臂（p）或長臂（q）位置。p來自法文petit (小)；q則是選定p的下一個字母，也有來自法文queue (尾巴)一說。
染色體帶經過適當染色之后，可於顯微鏡底下觀察。每個帶皆有編號，最靠近着絲粒（centromere）的為1號。
着絲粒
端粒