每日专业名词 | 通俗解释

 

2021年04月27日

碰到陌生pathway不要怕,查一下就行了。

membrane potential 膜电势 - 神经元之间的信号传导

Neuromuscular junction 肌肉神经接点

 

Single-cell isolation 单细胞分离技术【认真区分这些技术,不要一团浆糊】 

Serial dilution 连续稀释 - 拿不同的培养液连续稀释,得到单细胞悬浮液。优点:convenient operation, low cost,in vitro里应用广泛;缺点:time-consuming,无法精准得到target cells

Micromanipulation 显微操纵 - 直接在显微镜下人工分离,缺点:low throughput

fluorescence-activated cell sorting (FACS) 荧光激活细胞分选术 - 基于flow cytometry【刚科普过】,优点:高通量自动分选;缺点:requires a large number of cell suspensions,不适合分离low-abundance cells,mechanical damage

immunomagnetic separation (IMS) 免疫磁化分离 - magnetic beads磁珠结合到细胞表面的抗体,缺点:操作相对复杂,优点:tumor samples已经应用

laser capture microdissection (LCM) 激光捕获显微切割 - 直接在显微镜下用激光切割target部位,优点:cancer里广泛应用;缺点:high cost, low flux, lack of automation, and limited accuracy

microfluidic platforms 微流体平台 - 现在的商业化的标准单细胞分离技术,优点:污染少、试剂便宜;缺点:装置复杂,门槛较高

参考:Progress and applications of single-cell sequencing techniques 

 

2021年04月20日

Flow cytometry 流式细胞术

用特定的带荧光的抗体标记细胞,然后用类似单细胞的flow技术来一个一个检测细胞的信号,一般会选择两个维度,最后就画出了一个二维的散点图(颜色代表了密度density)。

对于我们的ENCC,则用了 HNK1 和 p75NTR 两个抗体

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:

  • 从混合细胞中筛选出目标细胞FACS;
  • 从相对细胞计数到绝对细胞计数
  • 从相对定量到绝对定量分析
  • 从单色到多色荧光分析
  • 从细胞膜成份到细胞内成份分析
  • 液体中可溶性成分的流式细胞分析
  • 分子表型分析

参考:

Flow Cytometry - YT

flow cytometry : basic principles - YT - 推荐

Expression of Ret-, p75(NTR)-, Phox2a-, Phox2b-, and tyrosine hydroxylase-immunoreactivity by undifferentiated neural crest-derived cells and different classes of enteric neurons in the embryonic mouse gut

 


 

immunocytochemistry ICC 免疫细胞化学 - 对象是细胞

immunohistochemistry IHC 免疫组织化学 - 对象是组织

Immunofluorescence IF 免疫荧光 - 泛指

都是用抗体来检测蛋白的丰度、分布和定位。abundance, distribution, and localization

非常常用的荧光染色剂及其标记物:

  • DAPI (blue) - nuclear acid
  • SOX10 - dividing ENS progenitor cells
  • HUC/D+ immature neurons
  • 5-HT+ mature neurons

参考:

ICC vs IHC vs IF – Do You Know the Difference?

 

2021年04月08日

双同源重组系统 - 周斌是该领域的大佬

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【这部分是细胞和发育生物学的核心技术手段,门槛极高,必须搞懂。】

Cre-loxP系统有lineage tracing的功能,只要表达过就会一直mark。而直接敲进报告基因则比较局限,只有表达的时候才会mark。

Cre-loxP的几个核心成分

Cre是recombinase enzymes重组酶,lox是序列位点locus,Cre可以切掉两个lox之间的DNA序列,这些DNA序列可以很灵活,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 报告基因,用法非常灵活,即可以研究特定调控序列的功能,也可以与目标基因耦合,研究基因功能。较常用的报告基因有黄/红/绿色荧光蛋白(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 动物模型,实验的材料,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.

参考:

Overview of the reporter genes and reporter mouse models

Reporter Knockin 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 原位杂交

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 免疫组织化学

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 染色体条带

【现在还在用,主要是保证历史延续性,让现在的人能看懂古人的研究】

用特定的染色剂处理后的染色体会出现明暗交替的区域,这就是条带【banding,条带,名词】。【染色技术是鼻祖,所以条带也是一个非常原始的发现,远远早于functional annotation】

染色体是细胞内具有遗传性质的遗传物质深度压缩形成的聚合体,易被碱性染料染成深色,所以叫染色体(由染色质组成)。(英语:chromosome;希腊语:chroma=颜色,希腊语:soma=体)

核小体是染色体结构的最基本单位。核小体的核心是由4对组织蛋白(H2A、H2B、H3和H4)各两个分子构成的扁球状8聚体。【与组蛋白修饰密切相关,为什么没有H1?】在相邻的连接线之间结合着一个第5种组蛋白(H1)的分子。从染色体的一级结构到四级结构,脱氧核糖核酸分子一共被压缩了7×6×40×5=8400倍。

1970年后陆续问世的各种显带技术对染色体的识别作出了很大贡献。中期染色体经过DNA变性、胰酶消化或荧光染色等处理,可出现沿纵轴排列的明暗相间的带纹。按照染色体上特征性的标志可将每一个臂从内到外分为若干区,每个区又可分为若干条带,每条带又再分为若干个亚带,例如“9q34.1”即表示9号染色体长臂第3区第4条带的第1个亚带。由于每条染色体带纹的数目和宽度是相对恒定的,根据带型的不同可识别每条染色体及其片段。

条带出现的本质原因?

 


 

locus 基因座

一个典型的基因座可能写成“6p21.3”,解释如下

6 = 6号染色体
p = 短臂
21 = 2区, 1带
3 = 子带3

染色体上的短臂(p)或长臂(q)位置。p来自法文petit (小);q则是选定p的下一个字母,也有来自法文queue (尾巴)一说。

染色体带经过适当染色之后,可于显微镜底下观察。每个带皆有编号,最靠近着丝粒(centromere)的为1号。

着丝粒

端粒

 

posted @ 2021-03-10 16:24  Life·Intelligence  阅读(53)  评论(0编辑  收藏
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