A stably transformed cell, a stably expressing cell line, refers to a cell line that is constructed based on a certain cell line that persists overexpressing or interferes with a particular gene. Lentivirus infection-drug screening method is a widely used method for constructing stable plants, which has the characteristics of high efficiency integration and wide target cells.

one. Preparation and preliminary experiment

• To determine cell line related information, you need to include the following

Note: To avoid cell death after lentivirus infection, it is important to ensure that the cells are free of mycoplasma contamination!

• Pre-experiment to determine the MOI value

• Review the literature to determine the MOI of lentivirus in the target cell line;
• Refer to the data obtained, design gradient experiments, and explore the optimal MOI.

• Pre-experiment determines the amount of drug used for screening:

• Check the lethal dose information of Puromycin/Blastincidin and other stable cell lines in target cell lines;
• Referring to the data obtained from the review, determine the concentration gradient of the three drugs (if there is no relevant information, the range of the drug concentration gradient needs to be increased, and the number is increased to 6);
• Day0 cells were plated in 6-well plates, making Day1 cell fusion approximately 90%;
• Day1 is added to the drug according to the drug gradient set in (2);
• Change the day4 and re-add the drug;
• Day7 observation, find the 100% of the lethality of the hole, the concentration of the drug used in the hole, that is, the drug screening concentration.

Appendix 1: Experience screening drug dosage

two. Screening and construction of stable plants

Note: The following experimental parameters are described as an example of a stable plant, and the experiment needs to consider whether there is a stable stable strain!

• Cell plating:

The cells were seeded in 6-well plates (4 wells), making the next day cell fusion approximately 70%

• Viral infection:

Calculate the lentivirus volume according to the MOI value determined by the pre-experiment, add lentivirus (add 2 μL of ADV-HR per well);

• Change the liquid:

The next day of lentivirus infection, the cells were exchanged for treatment;

• Observe the infection efficiency:

72 hours after infection, the infection efficiency was observed, and the efficiency should not be lower than 40%.

• filter:

• Polyclonal stable strain: After 72 hours of infection, the drug was added to the 6-well plate, and the drug was added every 2 days. Drug screening should be continued for at least 14 days until the proportion of fluorescent cells observed under the microscope is 100%.

Note: In the morning when the drug is added for the first time, the cell state is observed after 4-6 hours. If the cell death is too much, the fresh drug-free medium needs to be replaced.

Appendix 2: Polyclonal stable strain

The fluorescence of the polyclonal stable strain is usually strong and weak.

• Monoclonal stable strain: based on the acquisition of polyclonal stable strains

• Limited dilution method:

• Take 24 1.5 ml EP tubes and add 800 μl of complete medium to each tube;
• Digest the polyclonal stable strain with trypsin (90% confluency, 10 ml medium to terminate digestion), take 80 μl to the first EP tube, mix well

Note: 1000 microliters of gun tip should be used, do not over-suck when mixing, so as not to damage the cells.

• Take 80 microliters from the first EP tube to the second EP tube, mix well, and so on.
• The cell suspension in the EP tube was seeded in a 96-well plate at 100 μL per well;
• After overnight incubation, observe columns 12-24 and look for wells containing only 1 cell and mark them;
• After culturing for 3-4 weeks, the cells in the labeled wells are expanded, and then digested and passaged, which is a monoclonal stable cell.

Note: Do not change the fluid during the first week of culture, then change the fluid every 3-4 days.

• Flat picking

• Count 100 polyclonal stable cells and inoculate them in a 10 cm culture dish;

Note: Try to blow evenly to prevent cells from agglomerating.

• After overnight culture, observe and find the location of individual cells and mark them on the bottom of the plate;
• Culture and culture for 2 weeks;

Note: Do not change the fluid during the first week of culture, then change the fluid every 3-4 days.

• The cells to be labeled are expanded to a white spot visible to the naked eye. A little trypsin is taken from the tip of the pipette and slowly dropped to the cells. After the cells are digested, the cells are quickly blown and the cells under digest are transferred to a 96-well plate. Passage amplification, which is a monoclonal stable cell. It takes about 2-3 weeks.

Note: It is advisable to select 20 per plate. When digesting, it is necessary to follow the principle of digesting the edge first and then digesting the center to prevent mutual contamination between clones . Do not change the fluid during the first week of culture, then change the fluid every 3-4 days.

Appendix 3: Monoclonal stable strain

The fluorescence intensity of the monoclonal stable strains was basically the same.

Third, the common problems in the construction of stable plants

1. Mycoplasma contamination

Because mild mycoplasma contamination does not affect cell growth and proliferation, it is ignored by many laboratories. However, mycoplasma is prone to eruption after virus-infected cells, and there are a large number of cell debris, which even leads to cell death, leading to the failure of stable strain screening. We recommend that the mycoplasma contamination in cells and culture environments be excluded at the beginning of the stable plant construction.

2. Other issues

In addition to mycoplasma contamination, the following problems often occur in the construction of stable plants.

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