What Is Transfection?

Transfection is a process in which eukaryotic cells actively or passively introduce foreign DNA fragments under certain conditions to obtain a new phenotype. ^[1] In essence, there is no fundamental difference from transformation. Whether transfection or transformation, the key factor is to treat E. coli cells with calcium chloride to improve the permeability of the cell membrane, so that foreign DNA molecules can easily enter the interior of the cell. So, customarily, people often refer to transfection as generalized transformation. Conventional transfection techniques can be divided into two categories: transient transfection and stable transfection (permanent transfection).

Transfection

Transfection is a process in which eukaryotic cells actively or passively introduce foreign DNA fragments under certain conditions to obtain a new phenotype. ^[1]

Transfection is the process by which eukaryotic cells actively or passively introduce foreign DNA fragments to obtain a new phenotype.

Cell Transfection Experiment

Transfected cell screening

1. Determine the optimal concentration for antibiotic action:

Different cell lines have different sensitivities to various antibiotics, so a pre-test should be done before screening to determine the minimum concentration of antibiotics on the selected cells.

1) Inoculate 8 wells of cells in a 96-well plate or 24-well plate 24 hours in advance. It is advisable to grow the inoculum to a 25% monolayer the next day. Place in a CO2 incubator at 37 ° C overnight.

2) Change the culture medium to antibiotic-containing medium, and the antibiotic concentration will increase in a gradient (0, 50, 100, 200, 400, 600, 800, and 1000 g / ml).

3) For 10-14 days of culture, the concentration of most cell death is prevailing, generally 400-800 g / ml. When selecting stable expression clones, it can be appropriately increased by one level compared to this concentration. Half of the screening concentration is used when maintaining.

2. Transfection was performed as before.

3 72 hours after transfection, the transfected cells were passaged in a 6-well plate at a ratio of 1:10 and replaced with a selection medium containing the antibiotic concentration determined in the preliminary test. Single cells can be seen in the 6-well plate, and single cells can be divided and multiplied to form a single resistant colony by continuous culture. At this time, two methods can be used to select monoclonals.

1) Filter paper method: immerse trypsin with a sterilized 5x5mm filter paper, paste the filter paper on a single cell colony for 10-15 seconds, remove the filter paper with adhered cells and place it in a 24-well plate to continue pressure culture. The cells were grown in a 24-well plate and transferred to a 25cm flask, and then grown to a 75cm flask.

2) Limit dilution method: After digesting the cells, make a continuous 10-fold dilution (10-2-10-10), and add each diluted cell to a 96-well plate and culture. After 7-10 days, select a single The cloned wells were cloned again.

4. ELISA or Western blot to detect the expression of foreign proteins in monoclonal cells. Because the expression levels of different clones are different, multiple clones can be selected at the same time to select the clones with the highest expression and pass them on for conservation.

Transfection

Some cationic polymer gene transfection technologies have been introduced internationally, with a wide range of applicable hosts, easy operation, low cytotoxicity and transfection efficiency.

Transfection reagent

The high rate is favored by researchers. Among them, dendrimers and polyethylenimine (PEI) have the best transfection performance, but the dendrimer structure is not easy to be further modified, and its synthetic process is complicated. Polyethyleneimine is an organic macromolecule with a high cationic charge density. Two carbon atoms apart, that is, every "third atom is a protonated amino nitrogen atom, making the polymer network at any pH It can serve as an effective "protonsponge" body. This polycation can transfer various reporter genes into cells of various species, and its effect is better than that of lipid polyamide. After further modification, its Transfection performance is better than dendrimers, and it has low cytotoxicity. A large number of experiments have proved that PEI is a very promising gene therapy vector. At present, when designing more complex gene vectors, PEI is often used as the core component.

Transfection efficiency

The study of linear PEI (LinePEI, LPEI) and its derivatives as gene transfection vectors is earlier than branched PEI (BranchedPEI, BPEI). In the past, it was considered that the The cytotoxicity is low, which is conducive to cell localization. Therefore, the transfection efficiency should be higher than that of BPEI. However, recent studies have shown that the high degree of branching of BPEI is conducive to the formation of small transfection complexes, thereby improving the transfection efficiency, but at the same time, the cytotoxicity is also increased. The ultra-highly branched, flexible PEI derivative contains additional secondary and tertiary amine groups. It was found that this PEI has low toxicity but high transfection efficiency in dyeing experiments.

Some products use various branched and ultra-high branched small molecule PEI to crosslink with various crosslinkers containing degradable bonds under physiological conditions, and a series of highly branched degradable PEI derivatives are synthesized. Thing. The branched structure of the polymer makes it highly positively charged, so it is easy to efficiently encapsulate various DNA, RNA molecules and plasmids to form small nanoparticles, thereby improving the transfection efficiency. When the formed complex enters the cell, The degradable chemical bonds contained in the physiological conditions are hydrolyzed in the cell, so that the cross-linked polymer is broken down into non-cytotoxic small molecules PEI. The transfection reagent with such a structure can obtain high transfection efficiency and low cells in vitro application. Toxicity and its degradability are also of great significance for in vivo applications.

The latest transfection reagents are now made of nanomaterials, such as Engreen. The principle is that the molecule contains many amino groups, which will undergo protonation at physiological pH. These protonated amino groups can neutralize the negative charge on the surface of the DNA plasmid, so that DNA molecules are compressed into a relatively small volume of DNA particles from the stretched structure and wrapped in them to protect the DNA from nuclease degradation. The transfection complex mainly transfers DNA into cells through endocytosis to form endosomes. DNA is released from the inclusion bodies, enters the cytoplasm, and then enters the nucleus for transcription and expression. The transfection reagents produced by nanotechnology show unique properties in the nanometer scale with strong ability to protect DNA and low toxicity.