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Purifier Logic+ Class II, Type A2 Biosafety Cabinets

Company: Labconco
Catalog#: Purifier Logic+ Class II, Type A2
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Endogenous C-terminal Tagging by CRISPR/Cas9 in Trypanosoma cruzi
Author:
Date:
2017-05-20
[Abstract]  To achieve the C-terminal tagging of endogenous proteins in T. cruzi we use the Cas9/pTREX-n vector (Lander et al., 2015) to insert a specific tag sequence (3xHA or 3xc-Myc) at the 3’ end of a specific gene of interest (GOI). Chimeric sgRNA targeting the 3’ end of the GOI is PCR-amplified and cloned into Cas9/pTREX-n vector. Then a DNA donor molecule to induce DNA repair by homologous recombination is amplified. This donor sequence contains the tag sequence and a marker for antibiotic resistance, plus 100 bp homology arms corresponding to regions located right upstream of the stop codon and downstream of the Cas9 target site at the GOI locus. Vectors pMOTag23M (Oberholzer et al., 2006) or pMOHX1Tag4H (Lander et al., 2016b) are used as PCR templates for ... [摘要]  为了实现克氏锥虫内源蛋白的C末端标记,我们使用Cas9 / pTREX-n载体(Lander等,2015)在3'末端插入特异性标签序列(3xHA或3xc-Myc)特定的感兴趣基因(GOI)。将靶向GOI 3'末端的嵌合sgRNA进行PCR扩增,并克隆到Cas9 / pTREX-n载体中。然后扩增通过同源重组诱导DNA修复的DNA供体分子。该供体序列包含标签序列和抗生素抗性标记,加上对应于位于终止密码子右上游的区域和在GOI基因座的Cas9靶位点下游的100bp同源臂。载体pMOTag23M(Oberholzer等,2006)或pMOHX1Tag4H(Lander等,2016b)用作DNA供体扩增的PCR模板。用sgRNA / Cas9 / pTREX-n构建体和DNA供体盒共转染的Epimastigotes然后用抗生素培养5周以选择双重抗性寄生虫。最终通过PCR和Western印迹分析验证了内源基因标记。
【背景】自从CRISPR / ...

Isolation of Primary Breast Cancer Cells from HER2 Transgenic Mice
Author:
Date:
2016-10-05
[Abstract]  HER2 is a tyrosine kinase receptor, which is overexpressed in about 30% of breast cancer patients. Its overexpression leads to mammary tumorigenesis and increased invasion and metastasis (Slamon et al., 1987). HER2 transgenic mouse (FVB/N-MMTVneu mouse) is a well-established model of mammary tumor in human (Fantozzi and Christofori, 2006). Although in vivo models are excellent for assessing the influence of various factors, especially microenvironment, on development of breast cancer, a convenient and less costly way to study the underlying molecular events is utilizing cells derived from the model under evaluation. In order to explore the molecular mechanism by which HOXB7 inhibits initiation, but promotes metastasis of breast tumors, we generated mouse breast cancer ... [摘要]  HER2是酪氨酸激酶受体,其在约30%的乳腺癌患者中过表达。 其过表达导致乳腺肿瘤发生和增加的侵袭和转移(Slamon等人,1987)。 HER2转基因小鼠(FVB/N-MMTVneu小鼠)是人类乳腺肿瘤的良好建立的模型(Fantozzi和Christofori,2006)。 虽然体内模型对于评估各种因素,特别是微环境对乳腺癌发展的影响是极好的,但是一种方便且成本较低的研究基础分子事件的方法是利用来自模型的细胞 评价。 为了探索HOXB7抑制起始,但促进乳腺肿瘤转移的分子机制,我们从HER2转基因小鼠产生小鼠乳腺癌细胞系(Liu等人,2015)。 该方案可用于从具有其他遗传背景的小鼠产生乳腺癌细胞系。

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