| In vivo Bioluminescence Imaging of Luciferase-labeled Cancer Cells
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Author:
Date:
2016-03-20
[Abstract] Over the past decade, in vivo bioluminescent imaging has emerged as a non-invasive and sensitive tool for studying ongoing biological processes within living organisms (Contag et al., 1997; Contag et al., 1998). Based on the detection and quantitation of the photons produced by the oxidation of luciferin by luciferase enzymes (Harvey, 1927), this technique has proved to be particularly useful in analyzing cancerous cells and monitoring tumor growth (Edinger et al., 1999; Sweeney et al., 1999; Vidal et al., 2015), providing a cost-effective insight into how the disease progresses in vivo, without the need of serial sacrifice of animals. This protocol describes in detail the procedure of obtaining luciferase-tagged tumors in ...
[摘要] 在过去十年中，体内生物发光成像已经成为用于研究活生物体内正在进行的生物过程的非侵入性和敏感性工具（Contag等人，1997; Contag et al。，1998）。 基于通过荧光素酶氧化荧光素产生的光子的检测和定量（Harvey，1927），该技术已经证明在分析癌细胞和监测肿瘤生长中特别有用（Edinger等， ，1999; Sweeney等人，1999; Vidal等人，2015），提供了关于疾病在体内如何进展的成本效益的洞察 ，无需连续牺牲动物。 该协议详细描述了在免疫受损的小鼠中获得荧光素酶标记的肿瘤的程序，其可以通过使用IVIS光谱成像仪通过生物发光成像进行研究。
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| Preparation of Recombinant Galectin-3 for Cancer Studies
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Author:
Date:
2016-01-05
[Abstract] Galectin-3 is a member of a class of proteins termed Galectins, characterized by their ability to bind glycans containing β-galactose (Cummings and Liu, 2009). Galectin-3 binds preferentially to proteoglycans terminating with N-acetyllactosamine (LacNAc) chains (i.e., tandem repeats of galactose) (Newlaczyl and Yu, 2011). Galectin-3 is unique among the galectins in its chimeric structure. It shares a conserved carbohydrate recognition domain (CRD) with the other galectins, but has a long amino-terminal tail that is thought to be involved in protein aggregation. It can also form homodimers through its CRD (Cummings and Liu, 2009). Galectin-3 has been found to have diverse functions in tumorigenesis including: signaling, apoptosis inhibition, immune suppression, cell growth, and ...
[摘要] Galectin-3是称为Galectins的一类蛋白质的成员，其特征在于它们结合含有β-半乳糖的聚糖的能力（Cummings和Liu，2009）。 Galectin-3优先结合以N-乙酰乳糖胺（LacNAc）链（即半乳糖的串联重复）终止的蛋白聚糖（Newlaczyl和Yu，2011）。 Galectin-3在其嵌合结构中的半乳凝素中是独一无二的。它与其他半乳凝素共享保守的碳水化合物识别结构域（CRD），但具有被认为参与蛋白质聚集的长氨基末端尾部。它也可以通过CRD形成同型二聚体（Cummings和Liu，2009）。已经发现Galectin-3在肿瘤发生中具有多种功能，包括：信号转导，凋亡抑制，免疫抑制，细胞生长和转移等。 ...
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| Generating Isogenic Deletions (Knockouts) in Francisella tularensis, a Highly-infectious and Fastidious Gram-negative Bacterium
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Author:
Date:
2015-06-20
[Abstract] Generating bacterial gene deletion mutants, also known as knockouts (KOs), is a powerful tool to investigate individual gene functions. However, fastidious bacteria such as Francisella tularensis (F. tularensis) often are difficult to genetically manipulate. Indeed, many different approaches have been tested to generate F. tularensis mutants. First, Tn5-based EZ::TN transposons have been successfully used to generate transposon libraries in F. tularensis (Qin and Mann, 2006; Weiss et al., 2007). However, creating a comprehensive transposon library with saturating mutations can be laborious, screening for gene disruption requires high-throughput assays where known phenotypes can be measured, and transposons may not completely inactivate the gene ...
[摘要] 产生细菌基因缺失突变体，也称为敲除（KO），是研究个别基因功能的有力工具。然而，诸如土拉弗朗西斯氏菌（emosa Francisella tularensis）（土拉弗朗西斯氏菌）之类的顽固细菌通常难以进行遗传操作。实际上，已经测试了许多不同的方法来生成F。 tularensi 的突变体。首先，基于Tn5的EZ :: TN转座子已经成功地用于在F中产生转座子文库。土耳其病（Qin and Mann，2006; Weiss等人，2007）。然而，创建具有饱和突变的综合转座子文库可能是费力的，筛选基因破坏需要高通量测定法，其中可以测量已知的表型，转座子可能不会完全失活目标基因或可能改变下游基因表达。第二，II组内含子（也称为Targetron）已用于灭活F。 （Rodriguez et al。，2008; Rodriguez et al。，2009）。 Targetron通过在质粒编码的RNA和染色体DNA之间形成复合物，随后将II组内含子插入感兴趣的基因而发挥功能。 Targetron的主要优点是它不需要抗生素抗性标记。然而，如转座子所指出的，targetron基因插入可能不能消除所有基因功能或可能影响下游基因表达。第三，同源重组可用于用选择性标记（例如抗生素抗性标记）完全替代染色体靶基因。这种经典的遗传技术已经在许多F中使用。土耳其语研究（Ramakrishnan等人，2008; ...
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