| Heterochronic Pellet Assay to Test Cell-cell Communication in the Mouse Retina
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Author:
Date:
2017-02-05
[Abstract] All seven retinal cell types that make up the mature retina are generated from a common, multipotent pool of retinal progenitor cells (RPCs) (Wallace, 2011). One way that RPCs know when sufficient numbers of particular cell-types have been generated is through negative feedback signals, which are emitted by differentiated cells and must reach threshold levels to block additional differentiation of that cell type. A key assay to assess whether negative feedback signals are emitted by differentiated cells is a heterochronic pellet assay in which early stage RPCs are dissociated and labeled with BrdU, then mixed with a 20-fold excess of dissociated differentiated cells. The combined cells are then re-aggregated and cultured as a pellet on a membrane for 7-10 days in vitro. During ...
[摘要] 构成成熟视网膜的所有七种视网膜细胞类型都是由普通的多能视网膜祖细胞池（RPC）产生的（Wallace，2011）。已经产生足够数量的特定细胞类型的RPC知道的一种方式是通过负反馈信号，其由分化细胞发射并且必须达到阈值水平以阻止该细胞类型的额外分化。评估负反馈信号是否由分化细胞发出的关键测定是异源沉淀测定，其中早期RPC被解离并用BrdU标记，然后与20倍过量的解离的分化细胞混合。然后将组合的细胞再次聚集并在细胞膜上培养7-10天。在这段时间内，RPC将会分化，BrdU + RPC的命运可以使用细胞类型特异性标记进行评估。开发这种沉淀测定的研究人员最初表明，当两种细胞类型混合在一起时，新生儿RPC与胚胎RPC相比，在加速进度条件下产生杆（Watanabe和Raff，1990; Watanabe等，1997）。我们已经使用这种测定来证明我们发现作为视网膜神经节细胞（RGC）分化的负调节物的声刺猬（Shh）促进RPC增殖（Jensen和Wallace，1997; Ringuette等，2014）。最近我们修改了异质性沉淀测定法，以评估视网膜无长突细胞的反馈信号的作用，将转化生长因子β2（Tgfβ2）鉴定为负反馈信号，并将Pten作为Tgfβ2应答的调节剂（Ma et al。，2007 ; ...
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| Optogenetic Mapping of Synaptic Connections in Mouse Brain Slices to Define the Functional Connectome of Identified Neuronal Populations
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Author:
Date:
2017-01-05
[Abstract] Functional connectivity in a neural circuit is determined by the strength, incidence, and neurotransmitter nature of its connections (Chuhma, 2015). Using optogenetics the functional synaptic connections between an identified population of neurons and defined postsynaptic target neurons may be measured systematically in order to determine the functional connectome of that identified population. Here we describe the experimental protocol used to investigate the excitatory functional connectome of ventral midbrain dopamine neurons, mediated by glutamate cotransmission (Mingote et al., 2015). Dopamine neurons are made light sensitive by injecting an adeno-associated virus (AAV) encoding channelrhodopsin (ChR2) into the ventral midbrain of DATIREScre mice. The efficacy and ...
[摘要] 神经回路中的功能连通性由其连接的强度，发病率和神经递质特性决定（Chuhma，2015）。使用光遗传学，可以系统地测量识别的神经元群体和定义的突触后靶神经元之间的功能性突触连接，以便确定该识别群体的功能性连接群。这里我们描述了用于研究由谷氨酸共转播介导的腹侧中脑多巴胺神经元的兴奋性功能性连接体的实验方案（Mingote等，2015）。通过将编码通道视紫红质（ChR2）的腺相关病毒（AAV）注射到DATIREScre小鼠的腹侧中脑中，使多巴胺神经元变得光敏。多巴胺合成酶酪氨酸羟化酶的免疫荧光证实ChR2表达在多巴胺神经元中的功效和特异性。然后，切片膜片钳记录由接受多巴胺神经元投影的区域中的神经元产生，并且确定兴奋性连接的发生率和强度。所有接受多巴胺神经元投射的区域的连接发生率和强度的总结构成功能性连接体。
【背景】为了建立特定神经回路的功能，有必要确定解剖连接，解剖连接的映射及其功能连接，连接的强度，发病率和神经递质性质的映射。使用单因素限制的病毒性突触后追踪技术，可以描述包括多巴胺系统在内的神经回路的复杂解剖连接（Callaway and Luo，2015; ...
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| Telomere Dysfunction Induced Foci (TIF) Analysis
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Author:
Date:
2015-11-20
[Abstract] Telomerase maintains telomeric DNA in eukaryotes during early developments, ~90% of cancer cells and some proliferative stem like cells. Telomeric repeats at the end of chromosomes are associated with the shelterin complex. This complex consists of TRF1, TRF2, Rap1, TIN2, TPP1, POT1 which protect DNA from being recognized as DNA double-stranded breaks. Critically short telomeres or impaired shelterin proteins can cause telomere dysfunction, which eventually induces DNA damage responses at the telomeres. DNA damage responses can be identified by antibodies to 53BP1, gammaH2AX, Rad17, ATM, and Mre11. DNA damage foci at uncapped telomeres are referred to as Telomere dysfunction-Induced Foci (TIFs) (de Lange, 2005; Takai et al., 2003).
The TIF assay is based on the ...
[摘要] 端粒酶在早期发育期间在真核生物中维持端粒DNA，〜90％的癌细胞和一些增殖性干细胞。染色体末端的端粒重复与shelterin复合物相关。该复合物由TRF1，TRF2，Rap1，TIN2，TPP1，POT1组成，其保护DNA不被识别为DNA双链断裂。临界短的端粒或受损的遮蔽蛋白可引起端粒功能障碍，其最终在端粒诱导DNA损伤反应。 DNA损伤反应可以通过抗53BP1，gammaH2AX，Rad17，ATM和Mre11的抗体来鉴定。未封端的端粒的DNA损伤灶被称为端粒功能障碍诱导的Foci（TIF）（de Lange，2005; Takai等人，2003）。
TIF测定基于使用针对遮蔽蛋白如TRF2之一的抗体对抗DNA损伤标记物例如γ-H2AX和端粒的抗体的DNA损伤的共定位检测（Takai等人，2003; de Lange，2002; Karlseder等人，1999）。我们在这里描述的方法可以用于正常的人类和癌细胞。
其他常用的方法 - Telomere限制性片段（TRF）分析（Mender和Shay，2015b）和端粒重复扩增方案（TRAP） ...
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