Introduction and history of transfection
|26.6.2021||Posted by tactical33 under Advertising & Marketing|
Transfection refers to the process by which eukaryotic cells acquire new genetic markers due to the incorporation of foreign DNA. The development of DNA transfection technology has had a huge impact on modern molecular biology. Gene transfection technology is not only an important tool for studying transgene and gene expression, but also a key step in gene therapy at present. The ideal gene transfection reagent should have the following characteristics: efficient transfection; safe; low cytotoxicity; simple method; time-saving and economical.
Mammalian transfection technology was introduced in the late 1960s and early 1970s.
At present, the methods of introducing foreign DNA into mammalian cells can be roughly divided into two categories, namely, biological methods and physical and chemical methods. Commonly used in physical and chemical methods are calcium phosphate method, microinjection, electroporation, liposome-mediated transfection, and the recent gene delivery technology using polymer as a carrier. Biological methods mainly use viruses as vectors to introduce foreign DNA into cells through viral infection. Among them, retrovirus and adenovirus transfection systems are the most commonly used.
Among them, viral vectors are characterized by high integration efficiency and can allow long-term expression of foreign genes in host cells. The disadvantage is that they have potential safety risks. The electroporation method and the microinjection method are characterized by a high transfection rate. The disadvantage is that they require expensive equipment. The former damages cells greatly, and the latter requires injection of cell by cell when introducing DNA, which is not suitable for large numbers of cells Research needs.
Calcium phosphate can achieve high transfection efficiency in 293T and other cells under good transfection conditions. But calcium phosphate transfection has a prominent problem, which is very unstable, especially affected by the pH value. Every reagent used in the experiment must be carefully calibrated to ensure quality, because it even deviates from the optimal conditions by one-tenth Each PH may cause the failure of calcium phosphate transfection. Often even the most skilled transfection experimenter cannot guarantee the success of each calcium phosphate transfection. They may often be frustrated by the inexplicable transfection failure. At the same time, they often find that small-volume transfections, such as transfections below 6-well plates, are more effective. However, once you switch to a large dish, the transfection efficiency often drops quickly. Birth defects of calcium phosphate are often the main cause of experimental obstruction.
There have been numerous reports in the literature that liposomes themselves can participate in cell physiological activities and cause up-regulation or down-regulation of gene expression. Such as participating in the regulation of PKC (protein kinase C) pathway (Biochemistry. 1992 Sep 22; 31(37): 9025-30); such as inhibiting the activity of ATPase (Biochim Biophys Acta. 2008 Apr; 1777(4): 362-8) ; Such as interacting with the mitochondrial membrane (J Biol Chem. 1989 Jan 25;264(3):1508-15); transfection of siRNA causes off-target effects and so on. The magnitude of cytotoxicity often means the magnitude of the impact on the physiological activities of cells. These effects of liposomes are the root cause of cytotoxicity. This will cause serious interference to relevant research data and even affect the conclusions of the research. This has attracted the attention of many researchers.
People have been looking for transfection reagents that are more effective, less toxic and have little impact on research. Since the toxicity of lipid transfection reagents to cells is determined by its lipid properties, many researchers and biological companies have focused the development of new generation transfection reagents on non-liposomal polymers. Some excellent reagent products are on the market.
The gene delivery technology using high polymer as a carrier is becoming a research direction. However, the currently used cationic polymer transfection reagents still have the problems of low transfection efficiency and cytotoxicity. The latest high-molecular polymer transfection reagent is a nano-polymer transfection reagent, which has high efficiency, safety and low cytotoxicity due to the use of new technologies and new materials. The principle is: the molecule contains many amino groups, which will be protonated at physiological pH. These protonated amino groups can neutralize the negative charge on the surface of the DNA plasmid, so that the DNA molecule is compressed from a stretched structure into a relatively small DNA particle. And wrapped in it to protect DNA from nuclease degradation. The transfection complex mainly transfers DNA into the cell through endocytosis to form an endosome. The DNA is released from the endosome, enters the cytoplasm, and then enters the nucleus for transcription and expression. Transfection reagents produced by nanotechnology exhibit unique properties of strong binding and protection of DNA and low toxicity at the nanometer scale.
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