■ 基本信息
別名: | Plasmid #47107 |
啟動子: | CMV |
復制子: | pUC |
終止子: | bGH poly(A) signal |
質粒分類: | 哺乳細胞質粒；哺乳編輯質粒；哺乳Cas9質粒 |
質粒大小: | 9812bp |
質粒標簽: | N-3×Flag,N-NLS,C-VP64,C-HA |
原核抗性: | Amp |
真核抗性: | G418 |
克隆菌株: | Stbl3 |
培養(yǎng)條件: | 37℃，5%CO2 |
表達宿主: | 293T等哺乳細胞 |
誘導方式: | 無需誘導 |
5'測序引物:
| CMV-F(CGCAAATGGGCGGTAGGCGTG)
|
3'測序引物: | BGH-R（TAGAAGGCACAGTCGAGG） |
■ 質粒屬性
質粒宿主: | 哺乳細胞 |
質粒用途: | 基因編輯 |
片段類型: | CRISPR
|
片段物種: |
|
原核抗性: | Amp |
真核抗性: | G418
|
熒光標記: |
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■ 質粒簡介
pCDNA-dCas9-VP64質粒是CRISPR/Cas9系列質粒，復制子是f1 ori、ori和SV40 ori，質粒大小是9812bp，帶有Ampicillin氨芐青霉素抗性，可轉化進Stbl3中培養(yǎng)，培養(yǎng)條件是LB培養(yǎng)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.
■ 質粒圖譜
■ 質粒序列
質粒序列請下載：
ZK617pCDNA-dCas9-VP64哺乳編輯質粒.txt
