Classification and filter of transfection

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





    1. DEAE-dextran method

    DEAE-dextran is one of the first transfection reagents used in mammalian cells. DEAE-dextran is a cationic polymer, which combines with negatively charged nucleic acid and is taken up close to the cell membrane. It is transfected with DEAE-dextran. It has been successfully applied to the study of transient expression, but it is not very reliable for stable transfection.

    1. Calcium phosphate method

    The calcium phosphate method is a calcium phosphate co-precipitation transfection method. Because the reagents are easily available and cheap, it is widely used in transient transfection and stable transfection research. The DNA and calcium chloride are first mixed, and then added to PBS slowly. The DNA calcium phosphate precipitate is formed, and finally the suspension containing the precipitate is added to the cultured cells, and the DNA is taken up by the endocytosis of the cell membrane. Calcium phosphate also seems to protect foreign DNA from degradation by inhibiting nuclease activity in serum and cells.

    1. Artificial liposome method.

    The artificial liposome method uses cationic liposomes, which has high transfection efficiency. It can not only transfect cell lines that are not easily transfected by other chemical methods, but also transfect different lengths of chromosomes from oligonucleotides to artificial yeast. DNA, and RNA, and protein. In addition, liposome in vitro transfection is suitable for both transient expression and stable expression. Unlike the past, liposomes can also mediate the transfer of DNA and RNA into animals and humans for gene therapy. LipoFiterTM liposome transfection reagent (LipoFiterLiposomalTransfection Reagent) is a high-efficiency cationic liposome transfection reagent suitable for transfecting plasmids or other forms of nucleic acids and nucleic acid protein complexes into cultured eukaryotic cells. It can combine with negatively charged nucleic acid to form a complex. When the complex is close to the cell membrane, it is endocytosed into endosomes and enters the cytoplasm. Then the DNA complex is released into the nucleus. As for how DNA passes through the nuclear membrane, its The mechanism is not yet fully understood.


    Including: ①Microinjection ②Electroporation ③Gene gun, etc. Although microinjection is laborious, it is a very effective method to introduce nucleic acid into cells or nuclei. This method is commonly used to prepare transgenic animals, but it is not suitable for studies that require a large number of transfected cells. Electroporation is often used to transfect cells that are not easily transfected by conventional methods such as plant protoplasts. Electroporation relies on pulsed electric current to perforate the cell membrane to introduce nucleic acid into the cell. The efficiency of introduction is related to the intensity and duration of the pulse. The gene gun relies on high-speed particles carrying nucleic acid to introduce nucleic acid into the cell. This method is suitable for cultured nucleus in vivo cells.



    Transfected cell selection

    1. Determine the optimal concentration of antibiotic action:

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

    1) Inoculate 8 wells of cells in a 96-well plate or a 24-well plate 24 hours in advance. The appropriate amount of inoculation is to grow into a 25% monolayer the next day, and place it in a CO2 incubator at 37°C overnight.

    2) Change the culture medium to a medium containing antibiotics, and the antibiotic concentration increases in a gradient (0, 50, 100, 200, 400, 600, 800 and 1000μg/ml).

    3) Cultivation for 10-14 days is based on the death concentration of most cells, generally 400-800μg/ml. When selecting stable expression clones, the concentration can be appropriately increased by one level compared to the half of the selected concentration when maintaining.

    1. The transfection is carried out according to the previous steps.
    2. After 72 hours oftransfection, the transfected cells were passaged in a 6-well plate at a ratio of 1:10, and replaced with a selective medium containing the antibiotic concentration determined in the pre-test. A single cell can be seen in the 6-well plate, and it can be seen that the single cell divides and multiplies to form a single resistant colony. At this time, two methods can be used to select a single clone.

    1) Filter paper method: Dip a sterile 5x5mm filter paper with trypsin, stick the filter paper on the single cell colony for 10-15 seconds, take out the filter paper with cells attached and place it in a 24-well plate to continue pressure culture. Cells were transferred to 25cm culture flasks after being overgrown in the 24-well plate, and then transferred to 75cm culture flasks after being overgrown.

    2) Limiting dilution method: After digesting the cells, make a continuous 10-fold dilution (10-2-10-10), and add the cells of each dilution to a 96-well plate for culture. After 7-10 days, select a single The wells where the clones grew were cloned again.

    1. 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 clone with the highest expression to pass down and preserve the seed.


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