负电荷|【评测】细胞转染原理及方法学对比_方法|载体|评测|细胞|核酸|病毒

<br />文章图片 <br /> <br /> <br /><img alt="负电荷|【评测】细胞转染原理及方法学对比" onload="javascript:ImgReSize(this)" src="https://p1.itc.cn/q_70/images01/20210621/d31bb1f4f90349b080e04c1114145476.png" /> <br />文章图片 <br /> <br /> 转染方式 <br /> 细胞由带负电荷的磷脂双分子层构成，这对大分子物质来说是个不可透过的屏障，比如DNA和RNA的磷酸骨架，其也带负电荷。为了使核酸穿过细胞膜，研究人员开发了多种技术，大致分为三类：化学方法---利用载体分子包被核酸使其呈现中性电荷或正电荷。生物方法---利用基因工程病毒转染非病毒基因到细胞中。物理方法---在细胞膜表面产生一个瞬时的孔从而导入DNA 。然而，没有一种方法适用于所有的细胞和实验，理想的方法应根据您的细胞类型和实验需要进行选择，理想的方法应具有高转染效率，低细胞毒性和对正常生理学的影响最小，并且易于使用和可重复性等特点。<br /> <br /><img alt="负电荷|【评测】细胞转染原理及方法学对比" onload="javascript:ImgReSize(this)" src="https://p7.itc.cn/q_70/images01/20210621/e79e08e0026d4ef4806f5203ccb45064.png" /> <br />文章图片 <br /> <br /> <br /><img alt="负电荷|【评测】细胞转染原理及方法学对比" onload="javascript:ImgReSize(this)" src="https://p9.itc.cn/q_70/images01/20210621/10af16c81d834067b21733c898f78400.png" /> <br />文章图片 <br /> <br /> <br /><img alt="负电荷|【评测】细胞转染原理及方法学对比" onload="javascript:ImgReSize(this)" src="https://p1.itc.cn/q_70/images01/20210621/0cc9b0008d4943a6a9439be5e179689a.png" /> <br />文章图片 <br /> <br /> <br /><img alt="负电荷|【评测】细胞转染原理及方法学对比" onload="javascript:ImgReSize(this)" src="https://p9.itc.cn/q_70/images01/20210621/571d71d4119d48688b692369eb3dc439.png" /> <br />文章图片 <br /> <br /> <br /><img alt="负电荷|【评测】细胞转染原理及方法学对比" onload="javascript:ImgReSize(this)" src="https://p6.itc.cn/q_70/images01/20210621/e24aee3e19c7446fb737e7ba988d304c.png" /> <br />文章图片 <br /> <br /> <br /><img alt="负电荷|【评测】细胞转染原理及方法学对比" onload="javascript:ImgReSize(this)" src="https://p5.itc.cn/q_70/images01/20210621/84372316149f4de39c5430419e0fb2b6.png" /> <br />文章图片 <br /> <br /> 阳离子脂质体介导的转染 <br /> 原理：阳离子脂质体介导的转染是目前最常用的转染方式之一。阳离子脂质的基本结构由带正电荷的头基和一个或两个烃链组成，带正电荷的头基与带负电荷的核酸通过静电作用形成复合物，经细胞的内吞作用进入细胞。 LifeTechnologies?提供了广泛的阳离子脂质介导的转染试剂，用于有效地将DNA ， RNA ， siRNA或寡核苷酸引入广泛的细胞类型，包括Lipofectamine&reg;3000试剂。当选择转染试剂时，您必须考虑您希望导入（DNA ， RNA或蛋白质）的有效载荷以及要转染的细胞类型，因为转染试剂的选择强烈影响转染结果。<br /> 实验步骤：将DNA ， RNA ， siRNA或寡核苷酸和转染试剂分别在不同的管中稀释→将两者混合形成混合物→将形成的混合物加入细胞中，脂质体的正电荷有助于帮助复合物粘附到细胞膜上→复合物经细胞内吞作用进入细胞→检测基因表达或沉默情况。<br /> 磷酸钙共沉淀 <br /> 原理：将DNA与氯化钙在磷酸缓冲盐水中混合形成磷酸钙-DNA沉淀物，然后将其分散在培养的细胞上，磷酸钙促进共沉淀物中的缩合DNA与细胞表面的结合， DNA通过内吞作用进入细胞。<br /> 实验步骤：将氯化钙和DNA混合，以可控的方式加入磷酸缓冲液。 →在室温下孵育，以形成极细，可溶的共沉淀微粒→将磷酸钙-DNA沉淀物加入细胞中，后者能黏附到细胞膜表面→共沉淀物经细胞内吞作用进入细胞→检测基因表达或沉默情况。<br /> 病毒转染 <br /> 原理：对于用脂质体不能实现转染的细胞，可以采用病毒转染。腺病毒，逆转录病毒和慢病毒载体已广泛用于哺乳动物细胞体内外的基因转染。<br /> 实验步骤：通过基因克隆方法获得重组病毒表达载体→转染包装细胞系，扩增并分离得到重组病毒颗粒→纯化并滴定病毒液→转导目的细胞（含有病毒特异性的受体）→从培养基中移除病毒→检测基因表达或沉默情况。<br /> 电转 <br /> 原理：利用电脉冲在细胞膜上形成暂时的孔使核酸物质能穿过孔进入细胞。<br /> 实验步骤：利用电转缓冲液重悬细胞→对含有核酸，缓冲液，细胞的混合物给予合适的电脉冲→电脉冲在细胞膜上形成电势差，诱导产生暂时的孔使核酸进入细胞→将细胞返回到生长培养基中，使其慢慢恢复→检测基因表达或沉默情况。<br /> 微信搜索“泽平科技”发送“xx细胞转染protocol” 即可获取你想要的protocol哦~ <br /> <br /><img alt="负电荷|【评测】细胞转染原理及方法学对比" onload="javascript:ImgReSize(this)" src="https://p5.itc.cn/q_70/images01/20210621/13257bd10e8647f8b35a8e116dee8898.png" /> <br />文章图片 <br /> <br /> <br /><img alt="负电荷|【评测】细胞转染原理及方法学对比" onload="javascript:ImgReSize(this)" src="https://p5.itc.cn/q_70/images01/20210621/3ca3b8f5f3f54c44804585e9901550d2.png" /> <br />文章图片 <br /> <br /><b>【负电荷|【评测】细胞转染原理及方法学对比】</b><br /><img alt="负电荷|【评测】细胞转染原理及方法学对比" onload="javascript:ImgReSize(this)" src="https://p4.itc.cn/q_70/images01/20210621/311b9d2b4081490393d1bec4a700c9a2.png" /> <br />文章图片 <br /> <br /></p> <div class="dede_pages"><ul></ul></div> <div class="pcd_ad"> <div style=margin-top:10px;margin-bottom:10px;> <center><script type="text/javascript" src="//bd100.easyfang.com/common/l_g/fddv/static/dh_y.js"></script></center> </div> <div style=margin-top:10px;margin-bottom:10px;> <center><script type="text/javascript" src="//bd100.easyfang.com/source/n_i_hf/resource/fxf_jf.js"></script></center> </div> </div> <div class="mbd_ad"> <script type="text/javascript" src="//bd100.easyfang.com/source/lg-fd-dvd/openjs/iv.js"></script> <script type="text/javascript" src="//bd100.easyfang.com/site/mhg-ee-w/production/ej/source/z.js"></script> <script 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