Bioparticle-mediated DNA transfection assay
Bioparticle-mediated DNA transfection assay
The following scheme is based on methods developed by Horch et al. (1999), Sanford et al. (1993), publications from major gene gun manufacturers (US/EG Bulletins 1688 and 2087; Bio-Rad), and Steve Finkbeiner (University of California, San Francisco). Francisco), and methods established by Steve Finkbeiner (University of California, San Francisco). This experiment was derived from the next volume of the Molecular Cloning Laboratory Guide (3rd edition) by [US] J. Sambrook D.W. Russell.
Operation method
Bioparticle-mediated DNA transfection assay
Materials and Instruments
CaCl2 Ethanol Glycerol Spermidine Plasmid DNA Cell Growth Medium Move makings For more product details, please visit Aladdin Scientific website.
Gene gun Gold or tungsten particles Lens paper Microcentrifuge tubes Cells or tissues to be transfected
Buffers & Solutions
The composition of the storage solutions, buffers and reagents is shown in Appendix 1.
Dilute the storage solution to the desired concentration
CaCl2 (2.5mol/L)
Ethanol
An unopened bottle of pure ethanol. Ethanol is hygroscopic and absorbs moisture in air. In the following method. Washing with watery ethanol in steps 1, 2, and 3 will interfere with the efficient transfection of cells and tissues.
Glycerol (50% aqueous solution)
Autoclave.
Spermidine (0.1 mol/L)
Dissolve an appropriate amount of spermidine (alkali-free type) in deionized water and filter through a 0.22um filter to remove bacteria. Aliquot the solution into small portions and store at -20°C. Prepare a new storage solution every month.
Nucleic Acids and Oligonucleotides
Plasmid DNA
If it is the first time a new cell line or tissue is transfected with a gene gun, expression plasmids encoding appropriately tagged genes should be used to optimize transfection conditions. Examples include vectors expressing E.coli.β-galactosidase, green fluorescent protein, β-glucuronidase (plant) or selective markers such as neomycin resistance. Plasmids expressing the target gene or cDNA can be used after the experimental procedure is optimized.
During transfection of biological particles . Researchers are divided on whether plasmid DNA purified with various chromatographic resins containing different amounts of lipopolysaccharide can be used. Obviously. Even if there is a small amount of endotoxin/lipopolysaccharide contaminating the plasmid DNA, the transfection efficiency is reduced It is recommended that plasmid DNA be purified by centrifugation with CsCl-ethidium bromide (see Scheme 10 in Chapter 1). Solubilize the purified plasmid DNA with H2O to a final concentration of 1ug/ul.
Culture medium
Cell growth medium [complete medium with (optional) selective medium]
Specialty Equipment
Gene Gun
Typically uses the Biolistic PDS-1000/He Particle Delivery System, available from Bio-Rad, which consists of a bombardment chamber separated by a vacuum connected to a helium line. A pump capable of generating 5 inches of Hg is required Usually, household vacuum lines cannot be used for this experiment. A high-pressure (2400-2600 psi) helium tank (>9.999% purity) fixed to the bench or wall is connected to the apparatus only.
Gold or tungsten particles (microcarriers)
DNA for transfection of cells is transferred into cells by tungsten or gold particles with a diameter of 0.6um to 5um, the optimal particle diameter for a particular cell or tissue is determined empirically. The particles are purchased from a company (e.g., Bio-Rad or Sylvania) and the DNA envelope is prepared as described in the first step below.
Lens paper
Microcentrifuge tube (1.5 ml)
Use good quality centrifuge tubes. Some brands or batches of centrifuge tubes will over bind the gelatinous particles used as warheads in gene gun experiments.
Additional Reagents
Step 8 of this protocol may require reagents listed in Chapter 17, Protocol 7.
Cells and Tissues
Cells or tissues to be transfected
Wall-cultured cells of all species can be bombarded for transfection when grown to 50%-80% confluence. Suspension-grown plant cells are filtered through Buchner Filter Whatman No. 1 filter paper (7 cm diameter), and the collected cells are placed on sterile filter paper impregnated with hypertonic medium (see Sanford et al. 1993 for details).
Freshly dissected mammalian tissues were cut into pieces of approximately 400 µm and placed in Petri dishes prior to bombardment as described by McAllister et al. (1995).
Cultured bacteria and yeast were collected by centrifugation at mid- or late-log phase depending on the species and strain, resuspended in a small amount of hypertonic medium, and placed on a thin layer of agar ( 1x108-2x109 cells) on filter paper placed in Petri dishes prior to bombardment.
Methods
1. Preparation of tungsten or gold particles
a. Weigh 60 mg of tungsten or gold particles in a 1.5 ml centrifuge tube. b. Place the tungsten or gold particles in a 1.5 ml centrifuge tube.
b. Add 1 ml of 70% ethanol to the particles and shake continuously for 5 min at room temperature, and leave the tube on the bench for 15 min.
c. Collect the particles by centrifugation at high speed for 5 s.
d. Gently discard the supernatant and resuspend the pellet with 1 ml of sterilized water and shake for 1 min. leave the tube on the bench for 1 min.
e. Centrifuge at high speed for 5S to collect the particles.
f. Wash with water more than 3 times (follow steps d and e).
g. After the fourth wash, discard the supernatant and resuspend the particles with 1 min 50% glycerol.
The concentration of the washed particles should be 60 mg/ml and can be stored at room temperature for 1~2 weeks. Prolonged storage may oxidize and reduce transfection efficiency.
2. For every 6 dishes of cells or tissues, prepare one DNA-encapsulated particle as follows:
a. Aspirate 50 ul (approx. 3 mg) with continuous shaking of the Particle Reservoir.
b. Transfer to a centrifuge tube and add the following solution while shaking:
Plasmid DNA (~2.5ug) 2.5ul
2. 5mol/L-CaCl2 50ul
0.1mol/L spermidine 20ul
After adding the above reagents, shaking continuously for 3 min.
Continuous shaking is necessary to ensure that the DNA is uniformly encapsulated in the particles.
c. Place the centrifuge tube on the bench for 1 min to precipitate the particles, and centrifuge at high speed for 2s to collect the particles.
Prolonged centrifugation may cause the metal particles to clump together and reduce transfection efficiency.
d. Discard the supernatant and add 140ul of 70% ethanol to the precipitated particles. Discard 70% ethanol and add 140ul of 100% ethanol without moving the particles, discard the supernatant and add 50ul of ethanol.
e. Tap the side wall of the centrifuge tube to re-suspend the particles, and gently shake the tube for 2-3% of the time.
3. Fix a large slide to the metal support of the gene gun with the manufacturer's fixation device. Wash the slide twice with 6ul of ethanol and wipe it dry with lens paper after each wash.
4. Vibrate the precipitate from step 2 for 1 min, and while vibrating, aspirate 6ul of suspension (about 500ug of particles) and spread it quickly over an area of 1 cm from the center of the slide.
5. Repeat steps 3 and 4 until the desired slides are covered with particles. Allow the ethanol suspension containing the DNA-encapsulated particles to dry on the slide.
6. Place the slide on the gene gun and bombard the cell dish or tissue sheet according to the instructions.
7. when the air pressure returns to atmospheric pressure, remove the damaged cells or tissues and place them under appropriate culture conditions. Discard broken slides and repeat steps 6 and 7 until all dishes have been bombarded.
8. For stable transformation, proceed directly to step 9. For transient expression, assay the cells 24-96 h after bombardment using one of the following methods:
- If E.coli.β-galactosidase-expressing plasmid DNA is used, assay the enzyme activity in the cell lysate according to the procedure outlined in Scheme 7 in Chapter 17. Alternatively, follow the histochemical staining method described in the additional protocol in Scheme 1 of this chapter.
- If a green fluorescent protein expression vector is used, examine the cells with a microscope under 450-490 nm light.
- If β-glucuronidase-expressing plasmid DNA is used, detect β-glucuronidase activity as described in the following additional protocol: Histochemical staining of monolayers for identification of P-glucuronidase.
- If other gene products are expressed, the newly synthesized proteins are analyzed by in vivo metabolic markers by radioimmunoassay, immunoblotting, immunoprecipitation, or determination of the enzymatic activity of cell extracts.
To minimize differences in transfection efficiency between dishes, it is preferable to (1) transfect several dishes with each construct (2) incubate for 24 h and then digest the cells with trypsin; (3) pool the cells; and (4) respread the cells on several dishes.
9. Isolation of stable transfectants: After incubation with complete medium for 48-72 h, bombarded cells were transferred to selective medium. The concentration of selective reagents and the culture conditions depend on the cell type. 