| Antisense Oligonucleotide-mediated Knockdown in Mammary Tumor Organoids
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Author:
Date:
2017-08-20
[Abstract] Primary mammary tumor organoids grown in 3D are an excellent system to study tumor biology. They resemble the organization and physiology of native epithelia more closely than cancer cell lines grown in 2D, and additionally model interactions with the ECM (Boj et al., 2015; Clevers, 2016; Shamir and Ewald, 2014). Mammary tumor organoids are therefore a promising model system to identify and characterize novel drivers of breast cancer that would be unlikely to be identified using 2D cell lines. Antisense oligonucleotides can be used to efficiently and specifically knockdown target genes in the cell (Bennett et al., 2017). They can be taken up freely by organoids without the need for a transfection agent, making them a convenient tool for routine lab studies and screens.
[摘要] 在3D生长的原发性乳腺肿瘤组织是研究肿瘤生物学的优秀系统。 它们类似于天然上皮的组织和生理学，比2D生长的癌细胞系更为紧密，另外还与ECM的模型相互作用（Boj et al。，2015; Clevers，2016; Shamir and Ewald，2014）。 因此，乳腺肿瘤组织因子是一种有希望的模型系统，用于识别和表征不可能使用2D细胞系识别的乳腺癌的新型驱动因素。 反义寡核苷酸可用于有效和特异地敲低细胞中的靶基因（Bennett等，2017）。 它们可以被有机物自由摄取，而不需要转染剂，使其成为常规实验室研究和筛选的便捷工具。
【背景】乳腺癌是全世界妇女中最常见的恶性肿瘤，是妇女癌症死亡率的第二大原因（Siegel等，2017）。为了改善现有的治疗方案，确定和调查具有预防乳腺癌进展潜力的新分子靶标至关重要。我们应用RNA-seq来产生与正常乳腺上皮细胞相比在原发性乳腺肿瘤中失调的长非编码RNA（lncRNA）的综合目录，并将30个先前未表征的lncRNA作为乳腺肿瘤相关RNA（MaTARs）进行优先排序。为了功能评估MaTARs作为肿瘤进展的关键驱动因素，我们对3D乳腺肿瘤组织中的所有30个MaTARs进行了反义寡核苷酸（ASO）介导的敲低分析（Diermeier等，2016）。
ASO是短（20-mers），含有硫代磷酸酯修饰的核苷酸的单链DNA分子以及2'-ribose（5-10-5 ...
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| Visualization of Intracellular Tyrosinase Activity in vitro
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Author:
Date:
2016-04-20
[Abstract] Melanocytes produce the melanin pigments in melanosomes and these organelles protect the skin against harmful ultraviolet rays. Tyrosinase is the key cuproenzyme which initiates the pigment synthesis using its substrate amino acid tyrosine or L-DOPA (L-3, 4-dihydroxyphenylalanine). Moreover, the activity of tyrosinase directly correlates to the cellular pigmentation. Defects in tyrosinase transport to melanosomes or mutations in the enzyme or reduced intracellular copper levels result in loss of tyrosinase activity in melanosomes, commonly observed in albinism. Here, we describe a method to detect the intracellular activity of tyrosinase in mouse melanocytes. This protocol will visualize the active tyrosinase present in the intracellular vesicles or organelles including melanosomes.
[摘要] 黑素细胞在黑素体中产生黑色素，这些细胞器保护皮肤免受有害的紫外线。 酪氨酸酶是使用其底物氨基酸酪氨酸或L-DOPA（L-3,4-二羟基苯丙氨酸）引发颜料合成的关键铜酶蛋白酶。 此外，酪氨酸酶的活性与细胞色素沉着直接相关。 酪氨酸酶转运到黑素体中的缺陷或酶中的突变或降低的细胞内铜水平导致黑素体中酪氨酸酶活性的丧失，通常在白化病中观察到。 在这里，我们描述了一种方法来检测小鼠黑素细胞中酪氨酸酶的细胞内活性。 该协议将使存在于细胞内囊泡或细胞器（包括黑素体）中的活性酪氨酸酶可视化。
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| Three Dimensional Spheroid Co-culture Invasion Assay
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Author:
Date:
2015-01-05
[Abstract] The assay was developed to investigate the impact of stromal cells of different types (in our case breast cancer associated fibroblasts stably manipulated to modify expression of genes of interest) on the invasive capacity of epithelial cancer cells (in our case breast cancer cell lines) (Verghese et al., 2013). Typical two dimensional invasion assays do necessarily account for the presence of extracellular matrix that is present around the stromal and tumour cells in vivo and therefore cellular behaviour within these cultures may be non-physiological. This spheroid assay was developed to attempt to replicate more closely the environment that is present around breast cancer stromal and tumour cells in actual tumours (Verghese et al., 2013). Extra cellular ...
[摘要] 开发该测定以研究不同类型（在我们的情况下乳腺癌相关的成纤维细胞稳定操纵以修饰目的基因的表达）的基质细胞对上皮癌细胞（在我们的乳腺癌细胞系）的侵袭能力的影响（ Verghese ，，2013）。典型的二维侵袭测定确实需要考虑在体内基质和肿瘤细胞周围存在的细胞外基质的存在，因此这些培养物中的细胞行为可能是非生理的。开发这种球状体测定以尝试更密切地复制存在于实际肿瘤中的乳腺癌基质和肿瘤细胞周围的环境（Verghese等人，2013）。包括由胶原IV和胶原I组成的细胞外基质，并且成纤维细胞和上皮细胞被给予发展"生理"相互作用的机会（Verghese等人，2013; Hooper等人， ，2006）。该方法由Nowicki等人（2008）开发，并且在Verghese等人（2013）中使用它来公开数据。
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