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sukeharu金虫 (小有名气)
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[求助]
siRNA knockdown
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最近在看基因高除的文章，打算下面开始做这方面的内容。但是对文章中出现的质粒，载体什么的不是太了解。希望能得到大家的帮助，非常感谢。 下面是一篇文章，里面的质粒什么的来源，查了之后没有找到，不知是质粒名称的书写有问题，还是其它的，大家可否帮我看看都是什么（最好是有MAP图什么的），从哪里可以买到。 Sample Text The mU6 pro vector containing the mouse U6 promoter (36) or the pSilencer 1.0-U6 siRNA expression vector (Ambion, Austin, TX) was used for the construction of mouse and human Met siRNA expression plasmids. The siRNA target finder and design tool provided by Ambion was used for selecting the siRNA sequences (see Table 1 footnote). We selected four mouse, three canine, and three human candidate siRNA sequences from Met mRNA sequences (Table 1) to minimize possible nonspecific reactivity with target sequences. Moreover, these sequences also survived a BLAST search to exclude coding sequences with 17 or more contiguous base pairs of homology to other genes. The oligonucleotides that encode the Met mRNA 19-mer hairpin sequences were cloned into an expression vector plasmid (the BbsI and XbaI sites in the mU6 pro vector and the ApaI and EcoRI sites in the pSilencer 1.0-U6 vector) and tested for Met suppression activity in either mouse or human cells, respectively. Sample Text For the construction of recombinant adenoviruses, the AdEasy Adenoviral Vector System (human adenovirus serotype 5, or Ad5; Stratagene, La Jolla, CA) was used. First, we recloned the selected siRNA sequences with the U6 promoter into a pShuttle vector. As a mock vector, U6 promoter without the siRNA sequence was used. Then, pShuttle vectors containing siRNA sequences were linearized with PmeI and cotransformed with pAdEasy-1 into BJ5183 cells by electroporation. Positive (homologously recombined) clones were selected and confirmed by PacI digestion. Adenoviral DNA plasmids with the correct insert were transformed into TOP10-competent cells (Invitrogen, Carlsbad, CA) and amplified. The linearized adenoviral DNA was prepared by digesting the plasmid with PacI, after which it was transfected into the packaging cell line HEK293. Transfected cells were cultured for 7 days, and the virus was harvested. After repeating one more amplification cycle, large-scale amplification was done by with Cell Factory tissue culture plates (Nunclon, Nalge Nunc International, Rochester, NY). Purification of the virus was done either by CsCl ultracentrifugation or by PEG8000 precipitation. The titer of the virus was evaluated through endpoint dilution. Sample Text Cells at 75 to 80% confluency were infected with Ad Met siRNA diluted in a small volume of growth medium containing 10% fetal bovine serum at a multiplicity of infection (m.o.i.) of 10 to 100 for 4 hours at 37°C. After 4 hours, fresh complete growth medium was added, and the cells were cultured in a CO2 incubator at 37°C. After 2 to 4 days in culture, the adenovirus-infected cells were collected for Western blotting, proliferation assays, invasion assays, or morphologic analyses. |
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sukeharu
金虫 (小有名气)
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2楼2016-04-20 10:34:34
3楼2016-04-20 11:34:25
sukeharu
金虫 (小有名气)
- 应助: 2 (幼儿园)
- 金币: 1417.8
- 散金: 53
- 红花: 5
- 帖子: 257
- 在线: 85.1小时
- 虫号: 1460913
- 注册: 2011-10-25
- 专业: 功能与智能高分子
4楼2016-04-20 12:25:07
