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Get a gene, put it in a plasmid, get that plasmid expressed in a host, could even be a bacterial host,right?
把一个基因导入一个质粒,让质粒在宿主中表达,这个宿主甚至可以是细菌,对吧
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Well, I talked about the problem of using plasmids in gene therapy, that it's very difficult to get them into cells.
我们之前讨论过,基因治疗中运用质粒的问题,质粒本身极难进入细胞
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I apply ligase, and I've got the plasmid that I had before but now with my gene, colored green here,inserted.
我用连接酶,就能在质粒中把基因,这里绿色的部分,插进去
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The sources are: one,the plasmid vector that we've picked, and the second is these genes that for some reason we're interested in.
第一个是我们选择的质粒载体,第二个是我们感兴趣的基因
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If your gene goes in, you've interrupted the gene for antibiotic resistance and those new organisms aren't going to be resistant to antibiotic anymore.
如果基因进入了质粒,原本对抗生素有抗性的基因就被打断了,那些新细菌就不再对抗生素有抗性
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The first step in the process is to take our plasmid which we've selected, and to insert the gene that we want into it.
这个过程的第一步,是取得我们选择的质粒,把我们需要的基因插入到里面
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For a minute just assume that we can do this and I'm going to show you how to do it on the next slide.
我们暂时先假设基因已经插入到了质粒里,我会在下一张幻灯片告诉你如何做到
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Now the plasmid started to do its thing, which is replicate and the gene gets transcribed.
那么质粒就可以开始进行复制,基因得以转录
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It's a gene that's on the plasmid, and this particular gene confers a specific biological property to cells that have the plasmid and can use it properly.
它是质粒自带的基因,如果细胞能够获得这种质粒,并正确的利用它的话,这段基因就能赋予该细胞某种生物学特性
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How would I put a gene that I'm interested in into a plasmid?
怎么把我想要的基因整合到质粒上呢
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The second issue is that the gene vector, in this case the plasmid, has to be compatible with the cells that you're trying to express the gene in.
接下来的问题是如何选择合适的基因载体,在这里用到的,质粒,必须与要用来表达基因的,受体细胞匹配
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This is the slide that I left on last time and the idea here was to use plasmids, which I talked about last Thursday, to introduce genes into animals.
这是我上节课没讲完的一张幻灯片,这里的思路是利用,上周四所讲过的质粒,以将基因导入动物体内
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Took it out of the virus completely, cloned it into a plasmid, that plasmid was expressed in a foreign host, in this case it was expressed in yeast cells.
将基因从病毒中完全提取出来,并克隆制成质粒,该质粒在外来宿主中表达,此例中质粒在酵母细胞中表达
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Take a plasmid, cut it open, insert a gene that we want into the plasmid, and then put that plasmid in a host cell, and let the host replicate it.
取一个质粒,把它切开,把目标基因放进去,然后把这个质粒植入到宿主细胞,让宿主来进行复制
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If you selected the cells right than the only ones that have that resistance to Ampicillin are the ones that successfully got your plasmid and are using this Ampicillin resistant gene.
如果你选对了细胞,那么对氨苄青霉素有抗性的就是,成功得到了质粒并表达了基因的细胞
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You could imagine strategies where you have multiple resistance genes on a plasmid, resistance to Ampicillin, to Penicillin, to Erythromycin for example, and you design strategies for separating out which cells are carrying the plasmid that you're interested in.
若质粒上有多种抗性基因,你就有很多筛选方法,例如对氨苄青霉素,青霉素,红霉素的抗性,而且你可以设计方法分离出,含有你感兴趣质粒的细胞
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We're cloning a gene of interest to us by sort of hijacking that biological mechanism by putting our gene on this plasmid and so our gene gets copied many times, as the plasmid gets copied many times.
我们克隆感兴趣的基因的做法,这个生物机制有点像是绑架,我们把基因绑在质粒身上,随着质粒被多次地复制,我们的基因也一起被多次复制
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One of the ways that it's used is to take the fertilized egg from an animal, a mouse, usually it's a mouse and inject in a vector that contains a gene that is involved in human disease of some sort.
其中一种应用就是,从动物,一般是老鼠体内取出受精卵,注入某种质粒,这种质粒带有与某些人类疾病相关的基因
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I talked about just not using viruses but using plasmid DNA together with lipids and that's another strategy for gene therapy.
这一部分我们不仅讲了病毒的应用,也讲了利用脂质体包裹的质粒DNA,基因治疗还有其它的策略
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How did we put our gene fragments into this plasmid DNA in order to make multiple copies of it, or to clone the gene?
如何把基因片段整合到质粒DNA上,来获得大量拷贝,或者说来克隆基因呢
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The first step is to take the DNA fragments that we're interested in and put them into this vector by basically cutting open the double stranded DNA and inserting the gene that we like in the region where we've cut.
第一步是把我们感兴趣的DNA片段,整合到这个载体里,基本的方法是切开质粒这个双链DNA,把我们想要的基因插入切开的区域
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What we're going to do is take this plasmid, with its promoter gene construct in it and engineer it, to take out the lacZ gene, leaving in the promoter and putting in the protein that we want - the gene for the protein that we want.
我们要做的就是提取包含,它自己的启动子基因的质粒,并对其进行一些修改,剔除lacZ基因,留下启动子,把我们的目标蛋白植入--,我们生产目标蛋白的基因
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