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- 文献和实验
- 技术资料
- 库存：
大量
- 英文名：
pLV-U6-gRNA scaffold-EF1-NEO
- 保质期：
详询
- 供应商：
北京百奥创新科技有限公司
- 保存条件：
低温
- 规格：
10 μg at 0.5 μg/μL in TE
Description：CRISPR (clustered regularly interspaced short palindromic repeats) and CRISPR-associated protein 9 (Cas9) are components of the leading genome or gene editing system, CRISPR-Cas9. Since it was developed in 2012, this gene editing tool has revolutionized biology research, making it easier to study disease and enabling faster drug discovery. The detection of catalytically inactive Cas9 (dCas9) protein has greatly expanded this gene editing system to foster the study of gene expression and regulation in a wide range of organisms. This dCas9 protein lacks the ability to create DNA breaks in the target and serves as a DNA binding protein. When it fuses with a transcriptional repressor such as the Krüppel-associated box (KRAB) repressor or a transcriptional activator such as the tripartite fusion of three transcription activation domains: VP64, p65 and Rta (VPR), it can effectively regress or activate gene expression. To do this, a single guide RNA (sgRNA) of the gene of interest must be present. The Cas9-sgRNA complex binds to DNA sequences that are complementary to the sgRNA and causes a steric block that halts transcript elongation by RNA polymerase in CRISPRi systems. While in CRISPRa systems, the Cas9-sgRNA complex recruits transcription factors to increase desired gene expression.
Gene specific gRNA that usually targets the promoter region is cloned into one of the sgRNA expression vectors in all the sgRNA vectors, human U6 promoter drives the sgRNA expression. pLV-U6-gRNA scaffold-EF1-NEO sgRNA Lentiviral Vector expresses gRNA with the human U6 promoter and NEO with the EF1 promoter to allow for NEO selection of transduced cells.
Vector Information：This is a lentiviral expression vector that contains all the elements for efficient and high yield viral production. A ubiquitous EF1 promoter drives the expression of NEO to allow for the selection of transduced cells. This vector is used in combination with the Cas9-KRAB or the Cas9-VPR to repress or activate the target gene expression, respectively.
IMPORTANT NOTICE：Store the vial at -20°C immediately upon receipt.
Specifications:
| Product Name | pLV-U6-gRNA scaffold-EF1-NEO |
|---|---|
| Shipping Condition | Room Temperature - 2 Day Shipping |
| Storage and Stability | Store at -20°C immediately upon receipt. This product is stable for 6 months when stored as directed. |
| Quality Control | This plasmid is sequence verified. |
| Restricted Use | For Research Use Only. Not for use in diagnostic or therapeutic procedures. |
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文献和实验Characterization of a PGA-Based Scaffold for Use in a Tissue-Engineered Neo-Urinary Conduit
testing) and biological characterization (cell viability and proliferation) of a polyglycolic acid-based scaffold used to tissue engineer a Neo-Urinary Conduit™. Such methods are more broadly applicable to characterization of other neo-organ product
（如某些原代细胞）。 无标记：适合短期池化实验，但无法富集。 2. 启动子 gRNA 一般用 U6 启动子（Pol III）。要注意 U6 对连续 T 敏感（poly-T 会导致转录提前终止），这也是 gRNA 设计时的禁忌之一。 Cas9 常用 EF1α、CMV、CAG 等。CMV 在某些细胞系（尤其是小鼠细胞和一些原代细胞）中容易沉默，长期实验建议选 EF1α 或 CAG。 3. 滴度：物理滴度 ≠ 功能滴度 这是最重要的一个坑。 物理滴度：p24 ELISA 或 qPCR 测基因组拷贝
无效的序列。 GC 含量控制在 40%–60%：过高（>80%）容易形成二级结构，过低（<30%）结合不稳定。 避免连续 4 个以上 T：U6 启动子是 Pol III，连续的 T 会被识别为转录终止信号，导致 gRNA 被截断。 5' 端建议是 G：U6 启动子偏好 G 起始，若不是 G，可以在最前面额外加一个 G（注意这会改变 spacer 长度到 21 nt，需确认设计工具是否按此建模）。 避开 SNP 位点：靶区域内如果存在常见 SNP（尤其是 seed 区，靠近 PAM 的 8–12 nt
