pIREShyg3哺乳动物表达载体

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载体订购信息

产品编号
GS-1062
产品名称
pIREShyg3 哺乳动物表达载体
市场价格
￥ 1000
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载体基本信息

载体名称:
pIREShyg3
载体类型:
哺乳动物表达载体
克隆方法:
/
拷贝类型:
高
载体抗性:
Ampicillin (氨苄青霉素)
筛选标记:
Hygromycin
载体大小:
5752 bp 点击查看完整序列
启动子:
CMV
测序引物5’:
CMV-F: 5'-CGCAAATGGGCGGTAGGCGTG-3'
测序引物3’:
pIRES-R:5'-GCCCTAGATGCATGCTCG-3'
报告基因:
/

载体图谱展示

载体图谱

载体说明

描述

pIREShyg3 contains the internal ribosome entry site (IRES) of the encephalomyocarditis virus (ECMV), which permits the translation of two open reading frames from one messenger RNA (1–3). After selection with hygromycin B, nearly all surviving colonies will stably express the gene of interest, thus decreasing the need to screen large numbers of colonies to find functional clones. To select for cells that express high levels of the gene of interest, the selective pressure for antibiotic resistance was increased by shifting the hygromycin B phosphotransferase gene downstream to a less optimal position for translation as directed by the IRES sequence (3). By decreasing the level of expression of the antibiotic resistance marker, the selective pressure on the entire expression cassette is increased, resulting in selection for cells that express the entire transcript, including the gene of interest, at high levels. The expression cassette of pIREShyg3 contains the human cytomegalovirus (CMV) major immediate early promoter/enhancer followed by a multiple cloning site (MCS) that precedes stop codons in all three reading frames, a synthetic intron known to enhance the stability of the mRNA (4), the ECMV IRES followed by the hygromycin B phosphotransferase gene, and the polyadenylation signal from SV40. Ribosomes can enter the bicistronic mRNA at the 5' end to translate the gene of interest and at the ECMV IRES to translate the antibiotic resistance marker. ?

应用

When using the pIREShyg3 Vector, the antibiotic exerts selective pressure on the entire expression cassette; thus, a high dose of antibiotic will select for cells expressing a high level of the gene of interest. This selective pressure also ensures that the expression of the gene of interest will be stable over time in culture. Unless your expression experiments require a pure population of cells, you can use the pool of cells surviving selection instead of isolating and characterizing clonal cell lines. We recommend selecting mammalian cultures in 250–600 μg/ml Hygromycin B (Cat. No. 631309) depending on the cell line. Be sure to establish a kill curve for each cell line and each lot of Hygromycin B to determine optimal selection concentration.

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