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细胞转染对初接触细胞实验的科研人员而言,是一道难过的坎儿,细胞转染率低的话,你珍贵的细胞可能就只能扔掉了。

一、转染的途径大致可分为物理介导、化学介导和生物介导三类:

1.物理介导:电穿孔法、显微注射和基因枪属于通过物理方法将基因导入细胞的范例。

2.化学介导:方法很多,如经典的磷酸钙共沉淀法、脂质体转染方法、和多种阳离子物质介导的技术。

3.生物介导:方法有较为原始的原生质体转染,和现在比较多见的各种病毒介导的转染技术。

理想细胞转染方法,应该具有转染效率高、细胞毒性小等优点。病毒介导的转染技术,是目前转染效率zui高的方法,同时具有细胞毒性很低的优势。但是,病毒转染方法的准备程序复杂,常常对细胞类型有很强的选择性,在一般实验室中很难普及。其它物理和化学介导的转染方法,则各有其特点。

需要指出的一点,无论采用哪种转染技术,要获得最优的转染结果,可能都需要对转染条件进行优化。影响转染效率的因素很多,从细胞类型、细胞培养条件和细胞生长状态,到转染方法的操作细节,都需要考虑。


二、常规转染技术:

1.瞬时转染(transient transfection):外源DNA/RNA不整合到宿主染色体中,因此一个宿主细胞中可存在多个拷贝数,产生高水平表达,但通常只持续几天,多用于启动子和其他调控元件的分析。一般来说,超螺旋质粒DNA转染效率较高,在转染后24~72h小时内(依赖于各种不同的构建)分析结果,常常用到一些报告系统和荧光蛋白,β半乳糖苷酶等来帮助检测。

2.稳定转染(Stable transfected):外源DNA既可以整合到宿主细胞中,也可能作为一种游离体(episome)存在。尽管线性DNA比超螺旋DNA转入量低但整合率高。外源DNA整合到染色体中概率很小,大约1/104转染细胞能整合,通常需要通过一些选择性标记。如来氨丙基转移酶(APH;新霉素抗性基因),潮霉素磷酸转移酶(HPH),胸苷激酶(TK)等反复筛选,得到稳定转染的同源细胞系。

瞬时转染和稳定转染的比较


三、转染方式

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。


艾柏森提供细胞转染荧光蛋白实验

image.png

服务优势

艾柏森生物科技有限公司致力于基因、重组蛋白、抗体、噬菌体文库等生物制剂的研发,并致力于为客户提供一站式的CRO技术服务。

公司成立至今,以自主研发为核心,围绕大量专业技术人才,先后搭建了蛋白表达纯化平台、抗体定制平台、抗体工程平台、抗体药物平台、质量分析平台以及模式动物基因工程研究平台。以抗体药物研发一条链解决方案为基础,我们主要为客户提供重组蛋白表达生产、抗体制备、噬菌体库构建与筛选,单克隆抗体药物开发(抗体筛选、抗体测序、抗体分析、抗体人源化与亲和力成熟、表位作图、抗体工程改造与抗体药效学)、稳定细胞株构建与cGMP生产、细胞免疫(CAR-T/CAR-NK)、蛋白晶体学、蛋白质组学、生物信息学、药理毒理学等技术服务。


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