Transient transfection is one of the ways to introduce DNA into eukaryotic cells. In transient transfection, recombinant DNA is introduced into a highly infectious cell line to obtain temporary but high-level expression of the target gene. The transfected DNA does not need to be integrated into the host chromosome, the transfected cells can be harvested in a shorter time than stable transfection, and the expression of the target gene in the lysate can be detected。

Features

The transient expression method has many advantages:

Transient transfection is one of the ways to introduce DNA into eukaryotic cells. In transient transfection, recombinant DNA is introduced into a highly infectious cell line to obtain temporary but high-level expression of the target gene. The transfected DNA does not need to be integrated into the host chromosome, the transfected cells can be harvested in a shorter time than stable transfection, and the expression of the target gene in the lysate can be detected.

First, the operation is simple and the cycle is short. The traditional stable transformation system requires at least several months or even longer time from transformation to obtaining transgenic plants, and the transformation efficiency is relatively low. The transient expression system only needs 2 to 4 weeks, avoiding the tedious process of plant tissue culture (Kapila et al. al., 1997);

Second, the expression efficiency is high. When the single-stranded T-DNA enters the plant cell, not only the genes transformed into the plant genome can be expressed, but many free foreign genes that are not integrated into the plant genome can also be expressed with a higher copy number. Its expression is not affected by gene location and gene silencing, so the expression efficiency is higher, and it can reach several times the expression level of stable transformation (Janssenand Gardner, 1990; Kapila et al., 1997; Yang et al., 2000; Wroblewski et al ., 2005);

Third, it is safe and efficient. Stably expressed transgenic plants have food chain pollution, gene drift caused by pollen transmission and other transgenic safety issues (Twyan et al., 2003). Gene transfer and its expression in transient expression are an independent process and do not produce heritable offspring. Its biological safety is much higher, and the test results are more intuitive and reliable;

Fourth, there is no need to screen genes. Stable transgenic transformation usually requires preliminary screening of antibiotics or other screening genes. The transgenic plants also have relative transgenic safety issues. Transient expression does not require screening of genes, which reduces the unsafe factors caused by antibiotics in transgenes, and the vector can be smaller. , It is easier to transform larger target fragments and multiple genes;

Fifth, it can be performed on whole plants. When analyzing the interaction of exogenous gene expression and biotic or abiotic stress, it can more truly reflect the gene expression pattern in plants.

What should be paid attention to when using HEK293 cells for transient transfection?

First of all, it must be operated under aseptic conditions. Before starting the operation, you need to clean and set the parameters of the biological safety cabinet, that is, UV sterilization for 30 minutes, and the fan can be used after 10 minutes.

Second, preparations before transfection:

The HEK293F cells with a density of 0.3-0.35X106cells/ml were subcultured in 20ml medium and cultured at 36.5°C, 175rpm, and 5% CO2. After 3 days, when the cell density is about 2-3X106cells/ml, use the medium SMM 293 TI to dilute the cell density to 1X106cells/ml, and the volume of each bottle of cell fluid is 20mL, then screw the bottle tightly and put it in a shaker to continue the culture, 2-4 hours After that, transfection can be carried out.

Third, prepare transfection solution (1ml): The dosage of plasmid DNA and transfection reagent should be optimized according to specific experiments. Take about 800ul of 150 mM sterile NaCl solution to dilute 10 μg of DNA, mix it and place it on the workbench for 5 minutes; add about 50 μl of Sinofection transfection reagent to the DNA diluent, the final total volume of the transfection solution is 1 mL, mix well Place it behind the workbench for 10 minutes.

Fourth, add the transfection solution dropwise to the cell culture solution, shake well, screw the bottle tightly and put it back on the shaker (36.5°C, turn off 5% CO2, 175rpm). Add SMS 293-I feed solution (0.7 mL/bottle, you can find out the specific amount) after 20-24 hours of transfection, and then feed it for 6-10 days the next day.

The gene expression can be detected 48~72h after transfection. Such as the production of recombinant proteins and antibodies.

You can also perform simple operations: directly add DNA and Sinofection transfection reagent to the cell suspension for transfection. But it is necessary to optimize the ratio and dosage of DNA and transfection reagent, and choose the best ratio.

Since the details of each experiment may be different, it is recommended to do an optimization experiment before transfection to determine the best ratio of sinofection transfection reagent to DNA.

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