pDsRed2-C1哺乳荧光质粒

pDsRed2-C1哺乳荧光质粒

基本信息

启动子: CMV promoter

复制子: pUC ori,f1 ori

终止子: SV40 poly(A) signal

质粒分类: 哺乳系列质粒；哺乳荧光质粒；哺乳红色质粒

质粒大小: 4675bp

质粒标签: N-DsRed2

原核抗性: 卡那霉素Kan（50μg/ml）

筛选标记: 新霉素Neo/G418

克隆菌株: DH5α等大肠杆菌

培养条件: 37℃，有氧 LB

表达宿主: 293T等哺乳细胞

培养条件: 37℃，5CO2

诱导方式: 无须诱导，瞬时表达

5'测序引物: CMV-F（CGCAAATGGGCGGTAGGCGTG）

3'测序引物: Sv40-polyA-R (GAAATTTGTGATGCTATTGC)

备注: 哺乳细胞红色荧光表达质粒

质粒简介

        pDsRed2-C1编码DsRed2,一种DsRed变体，其设计用于更快的成熟和较低的非特异性聚集。衍生自Discosoma sp。红色荧光蛋白DsRed2，如其祖细胞DsRed1，含有一系列沉默的碱基对变化,对应于哺乳动物细胞中高表达的人类密码子使用偏好。除了这些变化外，DsRed2含有六个氨基酸取代：V105A，I161T和S197A，导致转染细胞系中红色荧光的出现更加快速;和R2A，K5E和K9T，其阻止蛋白质聚集。（DsRed2可能与DsRed1形成相同的四聚体结构。）在DsRed2组成型表达的哺乳动物细胞培养物中，转染后24小时内可通过荧光显微镜检测发红细胞。在表达DsRed1的细菌和哺乳动物细胞系统中经常观察到的蛋白质的大不溶性聚集体在表达DsRed2的生物体中显着降低。更快成熟，更可溶性的红色荧光蛋白也被宿主细胞耐受良好;用DsRed2转染的哺乳动物细胞培养物在测试的这些细胞系中没有显示出降低的存活力的明显迹象，表达DsRed2的细胞显示与非转染对照相同的形态和生长特征。     

       pDsRed2-C1 encodes DsRed2, a DsRed variant that has been engineered for faster maturation and lower non-specifc aggregation. Derived from the Discosoma sp. red fuorescent protein (drFP583; 1), DsRed2, like its progenitor DsRed1, contains a series of silent base-pair changes that correspond to human codon-usage preferences for high expression in mammalian cells (2). In addition to these changes, DsRed2 contains six amino acid substitutions: V105A, I161T, and S197A, which result in the more rapid appearance of red fuorescence in transfected cell lines; and R2A, K5E, and K9T, which prevent the protein from aggregating. (DsRed2 may, however, form the same tetrameric structure as DsRed1 [3].) In mammalian cell cultures when DsRed2 is expressed constitutively, red-emitting cells can be detected by fuorescence microscopy within 24 hours of transfection. Large insoluble aggregates of protein, often observed in bacterial and mammalian cell systems expressing DsRed1, are dramatically reduced in organisms expressing DsRed2. The faster-maturing, more soluble red fuorescent protein is also well tolerated by host cells; mammalian cell cultures transfected with DsRed2 show no obvious signs of reduced viability—in those cell lines tested, cells expressing DsRed2 display the same morphology (e.g., adherence, light-refraction) and growth characteristics as non-transfected controls.

        The multiple cloning site (MCS) in pDsRed2-C1 is positioned between the DsRed2 coding sequence and the SV40 polyadenylation signal (SV40 poly A). Genes cloned into the MCS will be expressed as fusions to the C-terminus of DsRed2 if they are in the same reading frame as DsRed2 and there are no intervening stop codons. A Kozak consensus translation initiation site upstream of DsRed2 increases the translation efficiency in eukaryotic cells (4). SV40 poly A signals downstream of the MCS direct proper processing of the 3' end of mRNA transcripts. The vector backbone also contains an SV40 origin for replication in mammalian cells expressing the SV40 T-antigen, a pUC origin of replication for propagation in E. coli, and an f1 origin for single-stranded DNA production. A neomycin resistance cassette (Neor ), consisting of the SV40 early promoter, the neomycin/kanamycin resistance gene of Tn5, and polyadenylation signals from the Herpes simplex virus thymidine kinase (HSV TK) gene, allows stably transfected eukaryotic cells to be selected using G418. A bacterial promoter upstream of this cassette expresses kanamycin resistance in E. coli.

       pDsRed2-C1 can be used to construct fusions to the C-terminus of DsRed2. If a fusion construct retains the fluorescent properties of the native DsRed2 protein, its expression can be monitored by flow cytometry and its localization in vivo can be determined by fluorescence microscopy. The target gene should be cloned into pDsRed2-C1 so that it is in frame with the DsRed2 coding sequences, with no intervening in-frame stop codons. The recombinant DsRed2 vector can be transfected into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (5). pDsRed2-C1 can also be used as a cotransfection marker; the unmodified vector will express DsRed2.

质粒图谱

pDsRed2-C1哺乳荧光质粒使用说明：

     1、收到质粒干粉后请先5000rpm离心1min，再加入20μl无菌水溶解质粒，室温放置1min；

     2、从-80℃冰箱中取出相应的感受态，置于冰盒上解冻，并做好标记；

     3、取2μl质粒加至100μl感受态中，冰浴30min；

     4、42℃热激90s，再冰浴2min；

     5、加入900μl无抗的LB液体培养基，180rpm震荡培养45min；

     6、6000rpm离心5min，仅留100ul上清混匀菌体沉淀；

     7、混匀后的菌液加至对应抗性的LB平板上，倒入适量玻璃珠，涂匀液体；

     8、将平板正向培养1h，再倒置培养12h~16h；

     9、挑取单克隆菌落至对应抗性的LB液体培养基中，震荡培养12h~16h，根据实验需要提取质粒。

pDsRed2-C1哺乳荧光质粒注意事项：

      1、如果您收到的是甘油菌种，请先四区划线，挑取单克隆培养。

      2、如果第二天转化平板长的过多，请将质粒按比例稀释后再转化。