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- Ybscience
- 中国/美国
- YB-0241
- 2025年07月15日
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- 详细信息
- 文献和实验
- 技术资料
- 保存条件：
-20℃
- 保质期：
6个月
- 英文名：
pSilencer 2.1-U6 Puro
- 库存：
大量
- 供应商：
钰博生物
- 规格：
1ug/5ug
载体基本信息
| 出品公司: | Ybscience |
|---|---|
| 载体名称: | pSilencer 2.1-U6 Puro |
| 质粒类型: | RNAi载体 |
| 高拷贝/低拷贝: | -- |
| 启动子: | U6 |
| 克隆方法: | -- |
| 载体大小: | 4455bp |
| 5' 测序引物及序列: | SV40 |
| 3' 测序引物及序列: | -- |
| 载体标签: | -- |
| 载体抗性: | 氨苄青霉素（Ampicillin） |
| 筛选标记: | Puromycin |
| 备注: | -- |
| 稳定性: | -- |
| 组成型: | 组成型 |
| 病毒/非病毒: | -- |
载体质粒图谱和多克隆位点信息
载体简介
This Ambion® vector is for the expression of siRNA. It has an antibiotic resistance gene (puromycin) to enable selection of transfected cells and features the human RNA polymerase III promoter (U6). Sufficient reagents are provided for 20 reactions. • Select transfected cells to enrich the population of cells expressing your siRNA • Eliminate the need to synthesize RNA oligonucleotides for RNAi experiments • Supplied linearized and ready for ligation Compensate for Low Transfection Efficiencies and Perform Long-Term Studies The use of mammalian siRNA expression vectors with antibiotic selectable markers conveys many benefits. Selectable markers can help compensate for poor plasmid transfection efficiencies seen with some cell lines. In these cases, only a fraction of the transfected cells express the siRNA, and reduction in target gene expression with even a potent siRNA can be difficult to detect. Use of a selectable marker and transient antibiotic selection permits only cells that have received the marker-containing plasmid to live in the presence of antibiotic. Thus, all of these cells should be exhibiting RNAi. Use of selectable markers also permits long-term gene silencing studies of cells that take up the siRNA expression vector. Changes in phenotype due to reduced gene expression that may not be readily apparent only a few days after transfection can be followed over a longer period of time. How siRNA Expression Vectors Work Vectors that express siRNAs within mammalian cells typically use an RNA polymerase III promoter to drive expression of a short hairpin RNA that mimics the structure of an siRNA. The insert that encodes this hairpin is designed to have two inverted repeats separated by a short spacer sequence. One inverted repeat is complementary to the mRNA to which the siRNA is targeted. A string of thymidines added to the 3' end serves as a pol III transcription termination site. Once inside the cell, the vector constitutively expresses the hairpin RNA. The hairpin RNA is processed into an siRNA, which induces RNAi of the target gene. Design of Vector Encoded siRNAs In general, the selection of an siRNA target site for vectors is the same as that used for designing siRNAs that will be introduced directly into cells, with the added caution that strings of four or more thymidine or adenosine residues should be avoided to reduce the possibility of premature termination of the transcript. The length of the inverted repeats that encode the stem of the putative hairpin, the order of the inverted repeats, the length and composition of the spacer sequence that encodes the loop of the hairpin, and the presence or absence of 5' overhangs can vary within certain parameters. It is recommended to use inserts that encode a hairpin with a 19-nucleotide stem and a specific 9-base loop sequence.
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文献和实验U6 vector, nucleotide overhangs to the EcoR I and Apa I restriction sites are added to the 5' and 3' end of the DNA oligonucleotides, respectively (Figure 2). In contrast, for cloning into the pSilencer 2.0-U6, 2.1-U6, 3.0-H1, or 3.1-H1 vectors
a 9-nucleotide spacer (TTCAAGAGA), although other spacers can be designed. 5-6 T's are added to the 3' end of the oligonucleotide. In addition, for cloning into the pSilencer 1.0-U6 vector, nucleotide overhangs to the Eco R I and Apa I restriction sites
1.常用载体分类：2,常用载体元件介绍：(1) 启动子：启动子是位于结构基因5'端上游的DNA序列，能活化RNA聚合酶，使之与模板DNA准确的结合并具有转录起始的特异性。启动子主要分为广谱启动子和特异性启动子。广谱启动子为在各种组织、细胞上广泛表达的启动子；特异性启动子为在特异性组织或者细胞类型上表达的启动子。常用广谱启动子有CAG，CMV，EF1a，PGK等，还包含启动RNA的III型启动子U6和H1启动子；常用特异性启动子如成熟神经元特异性启动子hSyn，投射神经元特异性启动
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