Transient transfectlon

Many cell types, including macrophages, are difficult to transfect, even using transient strategies. One important consequence is that the proportion of cells in a culture which receives the transgene is small (often less than 20%). Under these circumstances, the outcome measures do not fully represent the behaviour of the whole population. Indeed, measurement of the impact of a transfected gene on the behaviour of an endogenous gene is likely to be uninterpretable. This problem can be overcome by co-transfecting cells with multiple DNAs, at least one of which functions as a reporter gene by virtue of the sensitivity to the action of the transgene. Successfully transfected cells will receive copies of all DNAs and the outcome measurement (reporter gene product function) will thereby be restricted to transfected cells. One drawback of this latter property is that transfected cells take up large amounts of DNA and consequently express large amounts of the transgene product. While this has the advantage of increasing sensitivity, it may produce a non-physiological circumstance where the level of gene product markedly exceeds the amount present in normal cells. Transient transfections are also limited with respect to the number of different genes which can be transferred at one time. When concentrations of input DNA are too high, generalized cell toxicity may result, undermining the value of the experimental data.

Primary cells, including normal macrophages obtained from animals or humans, are most often limited to transient transfection only and that is often achieved with only low frequency. Thus long-term cultured cell lines of macrophage lineage are often requisite to successful application of gene transfer technologies in the analysis of macrophage gene expression. There are a wide variety of transfection strategies and many have been applied in mononuclear phagocytes, though with variable succcss in cach case. The most commonly reported methods include the use of DEAE dextran, clect rap oration, or liposomal preparations to achieve effective gene transfer. The liposomal reagents are the most recently developed tool for transfection procedures. Many of these are now available commercially and are highly efficient; their use should substantially expand the application of this technology to mononuclear phagocyte populations and other cell types. Protocol 4 describes one method for the preparation of plasmid DNA for use in transfection experiments; Protocols 5-7 then provide three different methods for transient transfection of macrophages using this supercoiled plasmid DNA, all of which have been used successfully to transfect macrophages.

Protocol 4

Purification of supercoiled plasmid DNA

Equipment and reagents

• 20-gauge and 22-gauge needles

• 15 ml conical polypropylene centrifuge tubes

• Heat-sealable tubes for ultra cent rifugation and sealing device (Ami con)

• Test-tube support stand and clamp to secure centrifuge tubes for sample retrieval

• Ehrlenmeyer flasks of appropriate size for bacterial culture

• 250 ml polypropylene centrifuge bottles with screw caps (Fisher Biotechnologies)

• Bacteria containing plasmid of choice for purification

• Glucose buffer: 50 mM glucose, 25 mM Tris-HCl pH 8.0,10 mM EDTA

• Isopropanol

• OptiPrep (60% iodixanol; Life Technologies)

• Terrific broth (TB) (1 litre): prepare one stock solution of 12 g tryptone, 24 g yeast extract, and 0.5 ml of 80% glycerol plus H-,0 to 900 ml, and one stock solution of 2.31 g KH2P04 (anhydrous) and 12.54 g K2HPO^ plus H20 to 100 ml. Autoclave both stocks, cool to 60 °C. and combine.

Method

1 Inoculate 250 ml TB with 1 ml of the bacterial culture in exponential phase of growth.

2 Incubate the culture for 16 h at 37 °C with aeration.

Protocol 4 continued

3 Centrifuge the 16 h culture fluid at 2000 g for 20 min at room temperature, using two 250 ml centrifuge bottles.

4 Remove and discard the supernatant fluid from each bottle.

5 Resuspend each of the cell pellets in 10 ml glucose buffer.

6 Add lysozyme to a final concentration of 5 mg/ml in each bottle.

7 Incubate at room temperature for 10 min.

8 Add 20 ml of a 1:1 mix of 0.2 M NaOH and 2% SDS to each bottle.

9 Swirl gently and incubate again at room temperature for 10 min.

10 Transfer the culture bottles to ice.

11 Add 15 ml of 3 M sodium acetate to each bottle and incubate for 10 min.

12 Centrifuge the samples at 25 000 g for 30 min at 4 °C.

13 Transfer each supernatant preparation to a fresh 250 ml bottle; if the supernatant is not clear, centrifuge again as in step 12 to ensure the purity of the final preparation,

14 Add 0.6 vol. of isopropanol to each sample.

15 Incubate at -20°C for at least 30 min.

16 Centrifuge samples at 20 000 g for 30 min at 4°C to pellet the DNA.

17 Wash each DNA pellet briefly by adding 20 ml of 70% ethanol and centrifuging as in step 16.

18 Carefully remove the ethanol wash solution by aspiration,

19 Allow the DNA pellets to diy briefly.

20 Resuspend the washed and dried DNA pellets in 10 ml TE buffer.

21 Add OptiPrep to a final concentration of 27% (v/v), and DAPI to 0.005% (v/v), to each sample.3

22 Aliquot samples into ultracentrifuge tubes, using a 5 ml syringe and 18-gauge needle, and heat-seal according to manufacturer's instructions (fill each tube completely; if additional volume is needed, use a solution of TE/27% OptiPrep/0.005% DAPI).

23 Centrifuge samples for 16-24 h at 300000-350000 g and 4°C.

24 Secure tubes for sample retrieval using a clamp mounted on a test-tube support stand.

25 Illuminate the tubes with long-wave ultraviolet light in order to visualize the band of supercoiled plasmid DNA."

26 Carefully puncture the top of each tube with a 22-gauge needle and leave the needle in place.

27 Retrieve the plasmid DNA by aspiration, using a 3 ml syringe and 20-gauge needled

28 Pool identical samples in 15 ml conical centrifuge tubes,

29 Add 0.5 vol. of 7.5 M ammonium acetate and 1 vol. of 100% ethanol, based on the volume of plasmid DNA retrieved, to each tube and mix by inversion.

31 Aliquot samples into 1.5 ml microcentrifuge tubes.

32 Centrifuge at 12000-15000 r.p.m. and 4°C in a microcentrifuge for 30 min (if no precipitate is visible) or 5 min (if precipitate is readily visible).

33 Resuspend pellets in an appropriate volume of TE buffer."

34 Measure A260 and A2B0 to determine the concentration and purity of plasmid samples/

8 This is easiest to execute in 5 ml increments; use fresh TE to adjust volumes as necessaiy. b Protective eyewear should be worn while using a UV light source, cTo aspirate plasmid DNA efficiently, insert the needle, bevel down, just below the band of plasmid DNA. Rotate the needle so the bevel faces upward, then extract the illuminated band by gently pulling on the syringe plunger.

d The volume of TE required to resuspend the pellets will vary with the individual plasmid, but generally will be at least 1 ml per 250 ml of original bacterial culture volume. e Assuming a spectrophotometer path length of 1 cm, the concentration of the plasmid in solution is equivalent to A260 x 50 x dilution factor. The A2S0:A2S0 ratio provides a measure of plasmid purity, and should be between 1.7-2.0.

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