■ 基本信息
别名: | Plasmid #47107 |
启动子: | CMV |
复制子: | pUC |
终止子: | bGH poly(A) signal |
质粒分类: | 哺乳细胞质粒；哺乳编辑质粒；哺乳Cas9质粒 |
质粒大小: | 9812bp |
质粒标签: | N-3×Flag,N-NLS,C-VP64,C-HA |
原核抗性: | Amp |
真核抗性: | G418 |
克隆菌株: | Stbl3 |
培养条件: | 37℃，5%CO2 |
表达宿主: | 293T等哺乳细胞 |
诱导方式: | 无需诱导 |
5'测序引物:
| CMV-F(CGCAAATGGGCGGTAGGCGTG)
|
3'测序引物: | BGH-R（TAGAAGGCACAGTCGAGG） |
■ 质粒属性
质粒宿主: | 哺乳细胞 |
质粒用途: | 基因编辑 |
片段类型: | CRISPR
|
片段物种: |
|
原核抗性: | Amp |
真核抗性: | G418
|
荧光标记: |
|
■ 质粒简介
pCDNA-dCas9-VP64质粒是CRISPR/Cas9系列质粒，复制子是f1 ori、ori和SV40 ori，质粒大小是9812bp，带有Ampicillin氨苄青霉素抗性，可转化进Stbl3中培养，培养条件是LB培养基，37℃。
As evidence that the dCas9-VP64–gRNA system can activate gene expression in other cell types, we transfected expression plasmids for dCas9-VP64 and the four gRNAs targeting Ascl1 into mouse embryonic fibroblasts. Because the gRNA target sites are conserved in the human ASCL1 and mouse Ascl1 promoters, we also observed activation of Ascl1 expression 4 d after transfection in MEFs treated with plasmids encoding dCas9-VP64 and the four gRNAs。gene activation by dCas9-VP64 and combinations of gRNAs can be very specific, this is not necessarily a surrogate for DNA-binding, and additional studies are necessary to define the role of the gRNA target sequence in determining genome-wide Cas9 activity in mammalian cells. The RNA-seq results for both IL1RN and HBG1-HBG2 showed moderate downregulation of the IL32 gene (false discovery rate < 0.03) in the samples treated with dCas9-VP64 and gRNA expression plasmids compared to control samples treated with only an empty expression plasmid. Because both the IL1RNtargetedandHBG1-HBG2–targeted samples were similarlyaffected, it is unlikely that this is the result of off-target dCas9-VP64 activity related to the identity of the target sequences. Weconfirmed these results by qRT-PCR for IL32, which showed thatthis downregulation is a general response to dCas9-VP64, evenin the absence of gRNAs.
■ 质粒图谱
■ 质粒序列
质粒序列请下载：
ZK617pCDNA-dCas9-VP64哺乳编辑质粒.txt
