Calcium phosphate-mediated transfection of high molecular weight genomic DNA in cells
Calcium phosphate-mediated transfection of high molecular weight genomic DNA in cells
The following method is a modification of the phosphotransformation method established by Graham and Van der Eb (1973), in which high molecular weight genomic DNA is used instead of plasmid DNA. this method is particularly useful for establishing stable cell lines carrying transfected genes complementary to host chromosomal mutations (Segeetal. 1984, Kingsley et al. 1986). This experiment was derived from the next volume of the Laboratory Guide to Molecular Cloning (3rd edition) by [American] J. Sambrook D.W. Russell.
Operation method
Calcium phosphate-mediated transfection of cells with high molecular weight genomic DNA
Materials and Instruments
Exponentially growing mammalian cell cultures Move makings For more product details, please visit Aladdin Scientific website.
CaCl2 Glycerol HEPES Salt buffer Isopropyl alcohol NaCl Genomic DNA Plasmids with selective markers Cell growth media
Polyethylene test tubes Shepherd's crook Tissue culture dishes
Buffers & Solutions
The composition of storage solutions, buffers and reagents is shown in Appendix 1.
Dilute the storage solution to the desired concentration.
CaCl2 (2mol/L)
Filter to remove bacteria and aliquot into 5 ml aliquots for freezing.
Glycerol (15% V/V) 1XHEPES salt buffer.
The HEPES salt solution is filtered for sterilization and 15% of the glycerol is added off-pressure before use.
HEPES Salt Buffer
21 mmol/LHEPES
0.7 mmol/LNa2HPO4
137 mmol/LNaCl
5 mmol/LKCl
6 mmol/L dextrose
Adjust pH7.10, filter to remove bacteria; freeze in 25-50 ml aliquots. Dissolve before transfection, take a small amount to measure pH, and re-adjust to pH7.10 if necessary.
Isopropanol
NaCl(3mol/L)
Filter and sterilize, store at room temperature.
Nucleic acids and oligonucleotides
Genomic DNA
Prepare high molecular weight DNA from appropriate cells as described in Scheme 3 in Chapter 6, and dilute the DNA with TE buffer (pH 7.6) to 100ug/m. Transfection requires approximately 20-25ug of DNA per 90 mm dish.
Before transfection, genomic DNA must be cut to 45-60kDa size (see steps 2 and 3). It is best to determine the conditions for cutting the DNA during the prelab as follows: cut 2 ml aliquots of high molecular weight DNA with a 22# gauge needle for different numbers of cuts (e.g., 3, 4, 5, or 6). Perform 0.6% (m/V) agarose gel electrophoresis and stain with ethidium bromide or SYBR gold. Labeling with monomers or dimers of linear phage λDNA to optimize other shoving steps requires that the DNA be sheared to the appropriate size in a rancid petri dish for step 9.
Plasmids with selective markers
Plasmids with selective markers are selected as described in steps 3 and 12.
Medium
Cell growth medium (complete and selective)
Specialty Equipment
Polyethylene tubes (12 ml)
Shepherd's crook (elbow pipette)
Siliconized glass pasteurized pipettes with hooked ends
Tissue culture dish (90 mm)
Cells and Tissues
Exponentially growing mammalian cell cultures
Methods
1. On the first day of the experiment, exponentially growing cells (e.g., CHO cells) were spread onto Petri dishes with appropriate serum-containing growth medium at a density of 5x105 cells per 90 mm dish, and the cells were incubated for 16 h at 37°C in an incubator with 5% CO2.
2. On the second day, the high molecular mass DNA is cut into fragments of 45kb to 60kb in size with a 22# gauge needle as determined in the prelab (see the Materials section for genomic DNA).
Transfect 20-25ug of genomic DNA per 90 mm of cultured blood.
3. Add 0.1 volume of 3 ml/L NaCl with 1 volume of isopropanol to precipitate the DNA. collect the DNA with an elbow pipette. introduce the precipitate along the wall of the tube into another tube containing HEPES buffer (lml of DNA per 12-15ug). gently swirl the tube at 37°C for 2 h to re-solubilize the DNA. make sure that all of the DNA has been solubilized before proceeding with the following experiments.
If cotransfection is selectively flagged (see note in step 12 below), add the appropriate plasmid solution to the genomic DNA to a final concentration of 0.5ug/ml.
4. Place 3 ml aliquots of sheared genomic DNA in 12 ml polyethylene tubes (each aliquot can be used to transfect 2 petri dishes).
Different cell lines have different transfection efficiencies, and different screening methods can also lead to differences in transfection efficiency. In general, about 15-20 dishes of CHO cells must be transfected to obtain 3-10 stable transfectants.
5. Gently rotate each aliquot of genomic DNA and add 120ul of 2mol/LCaCl2 dropwise to form calcium phosphate-DNA precipitates. Leave the tube at room temperature for 15-20 min, the solution will become cloudy, but no visible precipitate clumps will form.
6. Aspirate the medium from two Petri dishes (step 1), gently add 1.5 ml of calcium phosphate-DNA precipitate to each dish, carefully rotate the dish so that the precipitate covers the cell layer, and leave at room temperature for 20 min, rotating the dish once during this time.
7. Gently add 10 ml of pre-warmed (37°C) growth medium to each dish. Incubate the cells at 37°C for 6 h in a 5% CO2 incubator.
8. Repeat steps 5 to 7 until all cells in the dishes are covered with calcium phosphate-DNA precipitates.
9. After 6 h of incubation, observe the cells in each dish under a light microscope. A "peppery" precipitate can be seen adhering to the cells. The precipitate is not powdered or clumped.
Under the light microscope, you need to rely on experience to determine what is a "peppery" precipitate. If you see a powdery or lumpy precipitate at this point, terminate the experiment. Failure to form a "peppery" precipitate in this step or the turbidity of the solution in step 5 is due to incorrect pH of the HEPES buffer salt solution used, too long an incubation time in step 5, or an inappropriate concentration of CaCl2 or DNA.
10. In most cases, treatment with glycerol at this point will enhance transfection efficiency. Shock the cells with glycerol:
a. Aspirate the medium containing the calcium phosphate-DNA precipitate.
b. Add 3 ml of 1xHEPE-configured 15% glycerol per petri dish of cells preheated to 37°C. Incubate for no more than 3 min at room temperature.
Care should be taken that the glycerol in the HEPE buffer does not adhere to the cells for too long. The range of optimal times is very narrow, varying from cell line to cell line and from laboratory to laboratory, so treat only a few dishes at a time and take into account the time it takes to aspirate the glycerol buffer. Do not exceed the optimal incubation time. Time should be measured in seconds!
c. Aspirate off the glycerol HEPE buffer and quickly wash the dishes twice with 10 ml of pre-warmed medium.
d. Add 10 ml of pre-warmed growth medium and incubate for 12-15 h at 37°C with 5% CO2 .
11. Change the solution with 10 ml of freshly prepared growth medium. Continue to incubate overnight at 37°C in an incubator with 5% CO2.
12. At this point (day 4), the cells should look normal under the microscope. The cells can be digested with trypsin and re-cultured in selective medium for 2-3 weeks to form complementary and/or resistant clones. Change the medium every 2-3 days.
The length of the screening period, the density of resurfaced cells and the screening conditions depend on the complementation or screening mutation or gene. The density of resurfaced cells in this step should preferably be between 2. 5X105 and 1X106 cells per 90 mm dish. This parameter is based on the experience of spreading dishes with different numbers of cells (without transfection) and screening. Logically, the cell density should be as high as possible while at the same time effectively killing non-complementary or non-resistant cells.
Cotransfection (e.g., with a plasmid conferring host G418 resistance) can be used to distinguish transfectants from revertants. Some mutant cell lines have revertant frequencies as high as (i.e., 1 in a million cells). False-positive results are problematic as the frequency of transfection is usually 2X10-7 and the frequency of cotransfection is , 10-8. Coupling a selection marker (e.g. G418 resistance) to the mutation/gene to be screened for can eliminate false positives. See co-transfection in the information section for detailed instructions.
13. Individual clones are then cloned and propagated for use in the assay (see Jakby and PaStan l979 and Chapter 16, 16.18, step 7 for methods).
Cells are fixed in pre-cooled methanol for 15 min, then stained with 10% Giemsa for 15 min at room temperature, rinsed under running water so that the number of clones can be recorded.The Giemsa staining solution should be freshly reconstituted with phosphate buffer or water before use and filtered through Whatman No. 1 filter paper. 
