Experiments on stable transfection of mammalian cell genes
Experiments on stable transfection of mammalian cell genes
Analyzing the function of a gene often requires the formation of a mammalian cell line containing the stably integrated gene. In transfection, approximately 1/104 cells will stably integrate the exogenous source and a dominant (positive) selection marker can be used to isolate stable transformed cells. Use a cell line that can be efficiently transfected to ensure successful transfection. In addition, suitable controls should be included to ensure that the target gene is not cytotoxic.
Operation method
basic program
Materials and Instruments
Mammalian Cells Move 1. Make sure that the lineage to be transfected can grow in independent colonies. For adherent cells, inoculate approximately 100 cells in a 10 cm tissue culture dish and incubate for 10 to 12 days, changing the fluid every 4 days. The viable cell colonies were counted after methylene blue staining.2. Determine the conditions for the selection of the parent cells: Cells growing in confluent sheets on a petri dish are distributed in a ratio of not less than 1:15 in culture medium containing different drug concentrations. Cultivate for 10 days. Change the medium every 4 days with a suitable selection medium and check for viable cells.3. Determine the most efficient method of transfecting parent cells. Calcium phosphate or electroporation methods may be used. For calcium phosphate transfection, the mother cells are passaged 1:15 into complete culture 1 day prior to the addition of DNA.4. Transfect the parent cells with the target gene, the molar ratio of the plasmid containing the target gene to the plasmid containing the marker gene is not less than 5:1. Replace the plasmid containing the target gene with the stretcher DNA as a control, and perform several transfections separately. Caveat If a 5:1 ratio is used, it is not necessary to link the selection marker gene to the target gene before transfection: if the selection requires a large number of selective plasmids, sometimes there is no state to ensure a 5:1 ratio, so the selection marker gene and the target gene to be studied are constructed in the same plasmid. The gene under study can be cytotoxic if transfection with the gene containing the gene under study does not result in a clone, whereas a clone can be produced in the control containing it. For more product details, please visit Aladdin Scientific website.
Complete Culture Solution
Incubator Centrifuge Tubes
5. Cells were allowed to multiply 2 times under non-selective conditions. Distribute the cells 1:15 in the selection medium.6. Inoculate more than 5 dishes in the selection medium to get as many cell colonies as possible that can be picked and expanded into cell lines, change the medium with the selection medium every 4 days, incubate for 10~12 days, turn the dishes to the light at an angle to observe the cell clones therein, and one of the dishes can be stained to facilitate the calculation of the number of cell colonies. Large, well-grown cell colonies were selected.