■ 基本信息
別名: | Plasmid #47107 |
啟動(dòng)子: | CMV |
復(fù)制子: | pUC |
終止子: | bGH poly(A) signal |
質(zhì)粒分類: | 哺乳細(xì)胞質(zhì)粒；哺乳編輯質(zhì)粒；哺乳Cas9質(zhì)粒 |
質(zhì)粒大小: | 9812bp |
質(zhì)粒標(biāo)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(dá)宿主: | 293T等哺乳細(xì)胞 |
誘導(dǎo)方式: | 無需誘導(dǎo) |
5'測(cè)序引物:
| CMV-F(CGCAAATGGGCGGTAGGCGTG)
|
3'測(cè)序引物: | BGH-R（TAGAAGGCACAGTCGAGG） |
■ 質(zhì)粒屬性
質(zhì)粒宿主: | 哺乳細(xì)胞 |
質(zhì)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(biāo)記: |
|
■ 質(zhì)粒簡(jiǎn)介
pCDNA-dCas9-VP64質(zhì)粒是CRISPR/Cas9系列質(zhì)粒，復(fù)制子是f1 ori、ori和SV40 ori，質(zhì)粒大小是9812bp，帶有Ampicillin氨芐青霉素抗性，可轉(zhuǎn)化進(jìn)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(zhì)粒圖譜
■ 質(zhì)粒序列
質(zhì)粒序列請(qǐng)下載：
ZK617pCDNA-dCas9-VP64哺乳編輯質(zhì)粒.txt
