pCDH-CMV-MCS-EF1-copGFP-T2A-Puro.doc

载体名称:

出品公司：
pCDH-CMV-MCS-EF1-copGFP-T2A-Puro
SBI
质粒类型:
慢病毒表达载体；cDNA表达载体；双启动子载体
克隆方法:
多克隆位点，限制性内切酶
启动子:
CMV
载体大小:
--
5' 测序引物及序列:
CMV-F :CGCAAATGGGCGGTAGGCGTG
3' 测序引物及序列:
--
载体标签:
无
载体抗性:
氨苄青霉素（Ampicillin）
筛选标记:
GFP、Puromycin
克隆菌株:
E.coli cells(RecA-)推荐: Stbl2 ,OmniMAX 2 T1R
宿主细胞（系）:
常用细胞系，如HeLa, HEK293, HT1080, H1299
备注:
pCDH-CMV-MCS-EF1-copGFP-T2A-Puro慢病毒表达载体是基于HIV的慢病毒载体；
用于cDNA表达和克隆；高效转染细胞，建立稳定细胞系；
CMV启动子驱动目的基因的高水平表达，EF1a启动子驱动报告基因的中 等水平的表达。
产品目录号:
CD513B-1
稳定性:
稳表达
组成型/诱导型:
组成型
病毒/非病毒:
慢病毒（HIV)

载体质粒图谱和多克隆位点信息

pCDH 慢病毒载体的包装载体及细胞系:The expression lentivector contains the genetic elements responsible for packaging, transduction, stable integration of the viral expression construct into genomic DNA, and expression of the target gene sequence. The packaging vector provides all the proteins essential for transcription and packaging of an RNA copy of the expression construct into recombinant viral particles. To produce a high titer of viral particles, expression and packaging vectors are transiently co-transfected into producer mammalian cells (e.g., HEK 293 cells). For a detailed description of SBI’s Lentivector expression system,please refer to the Lentivector Expression System user manual.

启动子的选择：SBI provides a collection of cDNA cloning and expression vectors for various applications. A gene of interest can be cloned under a CMV or EF1 promoter with or without another expression cassette for a reporter gene (copGFP or PuroR). Genes can be either expressed transiently through transfection or stably expressed in a target cell line through transduction with packaged viral particles.
The major concern of cDNA expression in lentivectors is the efficiency level and stability of expression in target cell lines.
The Cytomegalovirus (CMV) promoter is a strong and most commonly used viral promoter that constitutively expresses downstream genes. While the CMV promoter works perfectly in the most common cell lines, it shows poor expression in some stem cell lines and hematopoietic cell lines (R.F. Doll, 1996; E.D. Papadakis, 2004).
The housekeeping elongation factor 1α (EF1) promoter has been shown to exceed and outlast CMV-mediated expression in retroviral, lentiviral, and adenoviral vectors, in hematopoietic cell lines (K. Tokushige 1997; H. Nakai, 1998; C. Teschendorf, 2002). EF1 also performs well in most common cell lines.
MSCV promoter is the 5’-LTR promoter of murine stem cell virus. When a portion of the U3 region of the 3’ HIV LTR was replaced with the U3 region of MSCV LTR, the resulted hybrid HIV/MSCV LTR has dramatically increased the transgene expression level in human CD34+ hematopoietic cells (J.K. Choi, 2001). After integration into genomic DNA, this promoter transcribes a long transcript with an intron in the 5’UTR flanked with splice donor and acceptor sites derived from the lentiviral vector. Further studies found that additional CpG mutations in the MSCV LTR reduced transcriptional silencing in embryonic stem cells (C.S. Swindle, 2004). We constructed cDNA expression vectors with the CpG-deficient MSCV incorporated into the 3’ HIV LTR. After integration into genomic DNA, 3’MSCV/LTR will replace the 5’LTR and provide a high level of expression of the target gene and reporter gene downstream.