Stable transfection by electroporation
Stable transfection by electroporation
This lab was derived from: Animal Cell Culture - A Guide to Basic Techniques (5th Edition)
Operation method
Scheme 27.12 Liposome-mediated transient transfection
Materials and Instruments
L8057-Y10 Cells D-PBSA Move Expression vector and DNA preparation: For more product details, please visit Aladdin Scientific website.
Culture Flasks Culture Plates F12 FB Medium G418(Invitrogen) Selective Medium Electroporation Electroporation Cup Electroporator II Electroporator-II Matching Performance Amplifier-Plus
pSVTKGH, linearized [ Selden et al., 1986]
This vector contains the SV 40 enhancer and the herpes simplex virus thymidine kinase promoter sequences, both of which initiate the expression of the human genotropic K hormone (hGH) gene. hGH can be secreted directly into the tissue culture medium and can be detected directly by radioimmunoassay [Ravid et al., 1991].
pcDNA3, linearized (Invitrogen ).
This expression vector contains the human cytomegalovirus enhancer-promoter upstream of the polyclonal site and downstream of the polyadenylate signal and transcriptional termination sequence of the bovine growth hormone (bGH) gene. pcDNA3 vector also contains the neomycin resistance gene, which is used as a screen for resistant clones that stably express the target gene. The pcDNA3 can also be used as a screening marker for cotransfection with reporter gene vectors as described in the methods in this chapter.
1. L8057-Y10 cells (F12/FB medium) were inoculated in 75 cm2 culture flasks at a concentration of 5X105 cells/ml (Ham's F12 containing glutamine (2 mmol/L ), penicillin (50 U/ml ) and streptomycin (5 μg/ml ) , with 10% FBS (heat inactivated; GIBCO # 16140-014)), and the cells were incubated at 37°C with 5% CO2.
2. At the late logarithmic growth stage, cells were collected and centrifuged at 4°C, 380 g for 5 min.
3. Wash the cell sediment with 10 ml D-PBSA and centrifuge at 380 g for 5 min at 4°C.
4. Wash the precipitate with 5 ml electroporation buffer and count the cells with a hemocytometer.
5. Centrifuge again at 380 g for 5 min at 4°C and collect the cells.
6. The cells were prepared into a cell suspension with a density of 1X106 cells/0.8 ml using electroporation buffer.
7. Transfer 0.8 ml of cells to a pre-cooled electroporation cup.
8. Add 50 μg and 5 μg of linear pSVTKGH and linear pcDNA3, respectively (if the purpose of the experiment is to detect the expression of a specific gene promoter-reporter gene, a separate electroporation cell without plasmid DNA is required as a negative control).
9. Hold the electroporation cup firmly with one hand and flick the bottom with the other hand to mix the DNA-cell suspension, and incubate the suspension on ice for 10 min.
10. bombard the cell suspension at 400V /500 μF and record the time.
11. Afterwards, remove the electroporation cup and set aside on ice for 10 min.
12. Transfer the bombarded cells to a centrifuge tube containing 10 ml of F12/FB, rinse the cup with a small amount of medium, return to the centrifuge tube, centrifuge, and collect the cells.
13. Re-suspend the cells in 20 ml of F12/FB and seed into 75 cm2 culture flasks and incubate at 37°C in a 5 % CO2 incubator to allow expression of the neomycin resistance gene.
14. After 24-48 h, the cells were collected by centrifugation and resuspended in 20 ml of selective medium.
15. Subsequently, fluid changes (selective medium) are required every 2-4 days to remove debris from dead cells and to allow for the growth of resistant cells, and the screening process takes at least two weeks.
16. Determine the expression of hGH in the cell supernatant (see section 27.11.5).