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DGKzeta基因方面几段+2009年12月28日
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Plasmids and DNA Constructs—Rat DGK-IV cDNA was a generous gift of K. Goto (Yamagata University School of Medicine, Sendai, Japan). pSREflagDGKIV was excised with EcoRI, and the 3.4-kb fragment encoding the DGK-IV cDNA was subcloned in the pcDNA3Myc vector (pCDNAMycDGK-IV). For expression experiments, all constructs were subcloned in the pEGFPBos vector in the GFP C terminus. For the MCA construct, pCDNA3MycDGKIV was digested with BstXI/BglII, and the 2.2-kb fragment was subcloned in pEGFPBos. To generate the ankyrin domains deletion (ANK), pCMV3MycDGKIV was SacI-digested, blunted, KpnI-digested, and subcloned in pEGFPBos digested with KpnI/SmaI. Site-directed mutagenesis was performed using the QuikChange site-directed mutagenesis kit (Stratagene). To generate the kinase-dead version of the enzyme, Gly-354 was replaced with Asp as described (25). We designed two mutations in the MARCKS homology domain; in the first we replaced Ser-259, Ser-266, Ser-271, and Ser-272 with Ala (mutant Ser 3 Ala), and in the second the same residues were replaced with Asp (mutant Ser 3 Asp). To mutate the CRDs, the first conserved His in each of the two CRDs of the protein, His-98 or His-173, were replaced with Gly either in the wild type protein or in the Ser 3 Asp mutant. The C-terminal domain (CT) construct, including the four ankyrin repeats and the PDZ-binding motif, was generated by PCR with appropriate primers, which included two restriction sites, SalI and XbaI. The PCR product was subcloned in the pGEM-T easy vector (Promega) and then excised with SalI/XbaI to be subcloned in the expression vector pEFBosCX-HA, which was previously digested with SalI/XbaI. Cell Lines and Transient Transfections—COS-7 cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 2 mM glutamine. For transfection, 60–80% confluent cells were transfected with LipofectAMINE (Invitrogen) according to the manufacturer’s instructions. After 24 h cells were harvested, and each sample was divided into two pellets for immunoblot and a DAG activity assay. The J-HM1-2.2 cell line was generated by stable transfection of the human muscarinic subtype 1 receptor in the Jurkat cell line (29). Cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum and 2 mM glutamine. Cells (1.2 107) were transfected by electroporation with 20 g of DNA using a Gene Pulser (Bio-Rad) at 270 V and a capacitance of 975 microfarads. Cells were immediately transferred to 30 ml of growth medium and assayed 24 h later. 希望大家支持，我不太懂基因方面，我想做dgk基因转染，但这方面我不会！！！谢谢大家我，我吧全部拿出，希望大家帮帮忙！！！万分感谢！！！ |
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sophia622
木虫 (小有名气)
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5楼2009-12-27 11:56:52
sophia622
木虫 (小有名气)
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★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★
lyh447lyh447(金币+16,VIP+0):特别感谢！！！ 12-27 00:08
lyh447lyh447(金币+16,VIP+0):特别感谢！！！ 12-27 00:08
| 质粒DNA构建——鼠DGK-IV的cDNA由K.Goto友情提供（Yamagata University School of Medicine, Sendai, Japan）pSREflagDgkIV用EcoRI切割，编码DGK-IV cDNA的3.4kb的片段亚克隆到pcDNA3Myc载体(pCDNAMycDGK-IV）。为了表达实验，所有构造被亚克隆到pEGFPBos载体GFP蛋白的C末端上；为了MCA的构建，用BstXI/BglII消化pCDNA3MycDGKIV，消化产生的2.2kb的片段亚克隆到pEGFPBos上。为了产生锚定蛋白结构域删除（( ANK）pCMV3MycDGKIV用SacI、KpnI进行双酶切，并且亚克隆到用KpnI/SmaI消化的pEGFPBos载体上。用QuikChange site-directed mutagenesis试剂盒(Stratagene)检测位点直接的突变。为了产生酶的激酶失活，Gly-354被Asp替代（25）。我们设计了2个突变在MARCKS同源结构域，第一个位点是Ser-259, Ser-266, Ser-271和Ser-272突变成Ala（突变Ser 3 Asp)，第二个相同的残基被Asp取代（突变Ser 3 Asp）。为了突变CRDs蛋白两个CRDs的第一个保守的His：His-98 或His-173在野生型中被Gly替代，在突变型中被Ser 3 Asp。C末端结构域构建包括锚定蛋白重复序列和PDZ结合基序，用合适的有SalI 和XbaI酶切位点的引物进行PCR扩增的方法产生。PCR的产物亚克隆到pGEM-T easy（Promega）载体中然后用SalI/XbaI酶切，然后亚克隆到用SalI/XbaI酶切的表达载体pEFBosCX-HA上。 |
2楼2009-12-26 21:08:07
sophia622
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lyh447lyh447(金币+10,VIP+0):非常感谢！！！ 12-27 00:08
zap65535(金币+3,VIP+0):助人为乐 12-27 01:58
lyh447lyh447(金币+10,VIP+0):非常感谢！！！ 12-27 00:08
zap65535(金币+3,VIP+0):助人为乐 12-27 01:58
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细胞系和瞬时转染——COS-7细胞系维持在加入胎牛血清和2mM谷氨酰胺的Dulbecco 改良 Eagle的培养基中，为了转染，60%-80%的融合细胞按照说明书用LipofectAMINE (Invitrogen)转染，24小时后收获细胞，每种样品分装成2个小管，分别做免疫沉淀实验和DAG活性测定。J-HM1-2.2细胞系是由Jurkat细胞系中人类亚型1毒蕈碱受体稳定转染产生（29）。产生的细胞保持在加有10%的胎牛血清和2mM谷氨酰胺的RPMI 1640培养基中。 20 g 的 DNA用270V和975电容的脉冲器电转化劲(1.2 107)细胞中，细胞迅速转移到30ml生长培养基中培养24h后测定 |
3楼2009-12-26 21:23:21
4楼2009-12-27 00:12:18