Protocol of Bacteria Culture and Gene Transfer

1. Growth of E. coli: Dissolve E. coli in 0.3 ml LB plus tetracycline (2 mg/ml) medium, transfer it into a tube containing 5 ml LB plus tetracycline (2 mg/ml) medium, 37 degree overnight, then freeze the E. coli (amplify in several tubes before freeze to get more samples).

LB Medium (Large-Bertani Medium) per liter: To 950 ml of deionized H₂O, add: bacto-tryptone 10 g, bacto-yeast extract 5 g, NaCl 10 g. Shake until the solutes have dissolved. Adjust to pH 7.0 with 5 N NaOH (about 0.2 ml). Adjust the volume of the solution to 1 liter with deionized H₂O. Sterilize by autoclaving for 20 minutes at 15 lb/sq.in. in liquid cycle.

2. Harvesting E. coli:

A. Streak an inoculum across one side of a plate using sterile technique. Resterilize an inoculating loop and streak a sample from the first streak across a fresh part of the plate, then incubate at 37 degree until colonies appear (overnight).

B. Transfer a single bacterial colony into 2 ml of LB medium containing tetracycline (2 mg/ml) in a loosely capped 15-ml tube. 37 degree overnight with vigorous shaking.

C. Pour 1.5 ml of the culture into a micro-centrifuge tube. Centrifuge at 12,000g for 30 seconds at 4 degree in a micro-centrifuge. Store the remainder of the culture at 4 degree.

D. Remove the medium by aspiration.

3. Lysis of E. coli:

A. Resuspend E. coli pellet in 0.1 ml of ice-cold Solution I (50 mM glucose, 25 mM Tris-Cl, pH 8.0, 10 mM EDTA, pH 8.0)

B. Add 0.2 ml of freshly prepared Solution II (0.2 N NaOH, 1% SDS), inverting the tube rapidly 5 times. Do not vortex. Store at 4 degree.

C. Add 0.15 ml ice-cold Solution III (5 M potassium acetate 60 ml, glacial acetic acid 11.5 ml, H₂O 28.5 ml), gently vortex, store on ice for 3-5 minutes.

D. Centrifuge at 12,000g for 5 minutes, 4 degree. Transfer the supernatant to a fresh tube.

E. Add 2 volumes of ethanol, mix by vortex, keep at room temperature for 2 minutes.

F. Centrifuge at 12,000g for 5 minutes at 4 degree.

G. Remove supernatant and any drops of fluid adhering to the walls of the tube.

H. Rinse the pellet of DNA with 1 ml of 70% ethanol at 4 degree, then remove supernatant and any drops of fluid adhering to the walls of the tube.

I. Redissolve the DNA in 0.05 ml of TE (pH 8.0) containing DNAase-free pancreatic RNAase (0.02 mg/ml). Vortex briefly. Store at 20 degree.

4. Purification of plasmid (1):

A. Transfer the DNA solution to a 15-ml Corex tube, and add 3 ml of an ice-cold solution of 5 M LiCl. Mix well, and then centrifuge at 10,000 rpm for 10 minutes at 4 degree.

B. Transfer the supernatant to a fresh 30-ml Corex tube. Add an equal volume of isopropanol. Mix well. Recover the precipitated DNA by centrifugation at 10,000 rpm for 10 minutes at room temperature.

C. Decant supernatant carefully, and invert the open tube to allow the last drops of supernatant to drain away. Rinse the pellet and the walls of the tube with 7% ethanol at room temperature. Drain off the ethanol entirely.

D. Dissolve the pellet in 0.5 ml of TE (pH 8.0) containing DNAase-free pancreatic RNAase (0.02 mg/ml). Transfer the solution to a micro-centrifuge and store at room temperature for 30 minutes.

E. Add 500 ml of 1.6 M NaCl containing 13% (w/v) polythylene glycol (PEG 800). Mix well. Recover the plasmid DNA by centrifugation at 12,000g for 5 minutes at 4 degree.

F. Remove supernatant. Dissolve the pellet of plasmid DNA in 0.4 ml of TE (pH 8.0). Extract the solution once with phenol, once with phenol:chloroform, and once with chloroform.

G. Transfer the aqueous phase to a fresh micro-centrifuge tube. Add 0.1 ml of 10 M ammonium acetate, and mix well. Add 2 volumes (~1 ml) of ethanol, and store at room temperature for 10 minutes. Recover the precipitated plasmid DNA by centrifugation at 12,000g for 5 minutes at 4 degree.

H. Remove the supernatant. Add 0.2 ml of 70% ethanol at 4 degree. Vortex briefly, and then centrifuge at 12,000g for 2 minutes at 4 degree.

I. Remove the supernatant, and store the open tube on the bench until the last visible traces of ethanol have been evaporated.

J. Dissolve the pellet in 0.5 ml of TE buffer (pH 8.0). Measure OD_260nm of a 1:100 dilution (in TE, pH 8.0) of the solution. Calculate the concentration of the plasmid DNA: 1 OD_260nm=0.05 mg of plasmid DNA/ml. Store the DNA in aliquots at -20 degree.

5. Transfer human interleukin 2 and swine growth hormone genes into human smooth muscle cells (HSMC):

A. Growth of HSMC: HSMC of aorta (from ATCC) is cultured in F12K medium containing 2 mM glutamine, 10 mM HEPES, 10 mM TES, 50 ng/ml ascorbic acid, 0.01 mg/ml insulin, 0.01 mg/ml transferrin, 10 ng/ml sodium selenite and 0.03 mg/ml endothelial cell growth supplement, FBS 10%.

B. Transfection: ~2´10⁷ cells suspended in 0.2 ml medium are seeded into a tissue culture chamber. 48-72 hours later, remove medium and add 0.2 ml fresh medium, then add 500 mg of plasmid in 0.05 ml calcium phosphate-HEPES-buffered saline, pH 7.0. Control the chamber temperature at 23 degree, 37 degree and 43 degree separately, or laser treatment. 4 hours (or other time length) later, change medium.

C. Detect: 12-48 hours after the addition of plasmid, measure the amount of interleukin 2 with immunology method in medium or in situ, or interleukin 2 gene amount with PCR in cell lysis solution or in situ.

6. Transfer interleukin 2 gene into rabbit, rat or mouse arteries (1): Fresh rabbit, rat or mouse arteries are fixed in a chamber with physiological buffer, then add plasmid solution and keep in different temperature (23 degree, 37 degree and 43 degree) or laser treatment. 12-48 hours after the addition of plasmid, measure the amount of interleukin 2 with immunological method in extract of artery or in situ, or interleukin 2 gene amount with PCR in artery extract or in situ.

7. Immunological measurement (2): Antibody of Anti-Interleukin 2 (human) could be gotten from Sigma. The assays could be done with the immunological method of western blotting or ELISA.

8. PCR in situ Hybridization (3): This measures the trace amount of the gene transfection and the tissue location of the transtection.

References:

1. J. Sambrook, E. F. Fritsch and T. Maniatis, Molecular Cloning, second edition, pp. 1.21-1.52 and 18.60-18.74, Cold Spring Harbor Laboratory Press, New York 1989

2. Frederick M. Ausubel, Roger Brent, Robert E. Kingston, David D. Moore, J. G. Seidman, John A. Smith and Kevin Struhl, Short Protocols in Molecular Biology, second edition, pp. 1.1-1.27, Greene Publishing Associates, New York 1992

3. Gerard J. Nuovo, PCR in situ Hybridization: Protocol and Applications, Second Edition, pp. 169-213, Raven Press, New York 1994

Protocol of RNA Extract

1. Suspend 10⁶-10⁷ cells (or equivalent amount of dispersed tissue) in PBS. Microcentrifuge 5 minutes, 10,000 rpm, 4 degree.

2. Resuspend cell pellet in 0.2 to 0.4 ml DEPC solution and vortex briefly. Microcentrifuge nuclei 10 seconds and transfer supernatant to a new tube. Incubate 20 minutes at 37 degree, then 10 min at 90 degree.

3. Microcentrifuge 5 min at 4 degree and transfer supernatant to a new tube. Use 0.005 to 0.01 ml directly in step 1 of basic protocol or ethanol precipitate and resuspend. Store frozen.

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File: Protocol of Gene Transfer.doc

4/21/1997-5/22/98