转染和基因编辑
转染是将核酸导入真核细胞的过程。细胞既可通过稳定转染将DNA整合到其基因组中，也可通过瞬时转染以进行蛋白的暂时表达。细胞转染结合化学、物理和生物学三种方法来研究细胞环境中的基因功能和表达。其应用包括基因治疗、诱导多能干细胞（iPSC）的产生、基于RNA干扰（RNAi）的基因沉默以及治疗性抗体和蛋白质的生产。
相关技术文章
- Transfection is the introduction of DNA, RNA, or proteins into eukaryotic cells and is used in research to study and modulate gene expression. Thus, transfection techniques and protocols serve as an analytical tool that facilitates the characterization of genetic functions, protein synthesis, cell growth and development.
- Browse our convenient transfection reagent selection guide to match the best reagent for your specific cell line and application needs.
- Automation is used for many applications to reduce variation caused by manual handling and to obtain reproducible results in high-throughput assays. High-throughput applications, such as knockdown studies or target screenings, often include cell transfection.
- Small inhibitory RNAs (siRNAs) have become the focus of interest in many laboratories. For the first time, these molecules offer an easy way to knock down the expression of selected genes in mammalian cells without having to resort to classical gene knockout techniques.
- Get tips for handling lentiviruses, optimizing experiment setup, titering lentivirus particles, and selecting helpful products for transduction.
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相关实验方案
- Our Universal Transfection Reagent is a unique formulation of a proprietary polymer blend used for transient and stable transfection of nucleic acids into various eukaryotic cell lines and hard-to-transfect primary cells. This is a fast and easy protocol is compatible with serum, serum-free medium and antibiotics.
- Protocols for Transfecting Common Cell Lines with X-tremeGENE™ Transfection Reagents
- Cell preparation for transfection Plate cells approx. 24 hours before transfection making sure cells are at optimal concentration (70 – 90 % confluency).
- Calcium phosphate transfection is a common method for the introduction of DNA into eukaryotic cells. This protocol can be optimized for use with a wide variety of cell types.
- Product manual provides detailed protocol for easy DNA transfection.
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常用转染方法
- 脂质和脂质体：阳离子脂质可形成包含DNA或RNA的脂质体以进行传递。这些脂质体可与细胞膜发生融合并将核酸释放进细胞中。
- 磷酸钙：磷酸钙可促进DNA与细胞表面的结合，使遗传物质通过内吞作用进入细胞。
- 阳离子聚合物：在基于聚合物的转染中，外源DNA可与阳离子聚合物，如聚乙烯亚胺（PEI）形成复合物，并通过内吞作用进入宿主细胞。
- 慢病毒转导：细胞会感染被修饰的慢病毒载体，而慢病毒载体会将其病毒RNA转化为双链DNA，进而整合到宿主基因组中进行传递。
- 微注射：首先把目标细胞置于显微镜下，然后使用细玻璃毛细管针将核酸直接注入细胞质或细胞核中。
- 电穿孔转染：细胞在高强度电流中使细胞膜不稳定，此时基因传递的通透性更强。
转染通常用于基因编辑和基因沉默技术，有助于人们对复杂生物过程的理解，进而利用基因疗法来治疗疾病。
- CRISPR-Cas系统基于细菌的防御机制，利用遗传CRISPR（聚簇有规律间隔的短回文重复序列）结合Cas（CRISPR相关）核酸内切酶在靶标位置切割基因组DNA并在体内去除或替换基因。
- 工程化的锌指核酸酶（ZFN）由DNA结合域和核酸内切酶构成，可在靶标位点切割DNA以进行基因编辑。
- 短发夹RNA（shRNA）和小干扰RNA（siRNA）等RNAi试剂通过抑制转录或激活序列特异性RNA降解过程来限制基因的转录水平以实现基因沉默。
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