Preparation of Recombinant Plasmid DNA for DNA-Mediated Gene llransfer R&my A&in, Michael WeinfeZd, and Malcolm C. Paterson 1. Introduction Recombinant plasmid constructs are frequently employed in transfec- tion experiments. With the availability of a wide spectrum of specialized and versatile eucaryotic cloning/expression vectors, investigators have been given powerful tools to expedite the elucidation of mechanisms governing gene expression, and to facilitate the identification of genes participating in diverse cellular processes (namely, metabolism, the immune response, diE- ferentiation and development, the repair of DNA damage, and malignant transformation). The success of a particular gene transfer experiment depends largely on the quality of the donor DNA preparation. Indeed, the requirement for highly purified form I (covalently closed circular supercoiled) plasmid molecules, to ensure both consistency and optimal levels of gene expression in “tran- sient” assays, as well as reproducible frequencies of stable transfection, is well documented. Many plasmid purification schemes exist, but the Triton lysis/CsCl “double banding” procedure is the one most frequently quoted as providing transfectiongrade DNA. The technique, however, does require a consider- able amount of time (3-5 d) and relies on specialized ultracentrifugation From: Methods in Molecular Biology, Vol. 7: Gene Transfer and Expression Protocols Edited by: E. J. Murray 0 1991 The Humana Press Inc., Clifton, NJ 3 Aubin, Weinfeld, and Paterson equipment. As a result, only a limited number of large-scale plasmid prepa- rations can be processed at once. A detailed description of this protocol was recently published by Gorman (I) and is presented here with only mi- nor modifications. By way of an alternative, this chapter also describes how milligram quan- tities of comparably pure form I plasmid DNA (i.e., free of contaminating bacterial chromosomal DNA, RNA, and other host cell components) can be obtained by incorporating the acidified-phenol extraction scheme of Zasloff and coworkers (2) as a final step to the preparative alkaline lysis procedure of Birnboim (3). The method is rapid (l-2 d) and lends itself to the preparation of multiple plasmid stocks. Briefly, plasmid molecules are first extracted from bacteria at alkaline pH (in the range of 12.0-12.6) in the presence of deter- gent. Under these conditions, linear (chromosomal) DNA will denature, whereas intact plasmid duplexes will not. Neutralization of the lysate at high ionic strength allows essentially all of the chromosomal DNA and most of the host cellular RNA and protein to precipitate, while the plasmid remains in solution. Complete removal of residual RNA and protein is then accomplished through a sequence of LiCl precipitation, RNAse and proteinase K digestion, phenol extractions, and ethanol precipitations. At this stage, a plasmid preparation consisting of >90% form I molecules is routinely obtained. Fx- traction with acidified phenol is then used to remove selectively linear and open circular duplexes from the preparation in one step. The mechanism appears to exploit differences in the hydrophilic character of the different DNA species (4). At pH 4.0 in the presence of 75 mMNaC1, open circular and linear molecules are converted to their single-stranded forms and are there- fore less hydrophilic than the form I duplexes, because the bases are not shielded from the aqueous environment. In addition, the protonation of the nucleotide bases contributes to reduce significantly the overall charge of the DNA. As a result, open circular and linear DNA species are partitioned to the acidified phenolic phase, whereas intact form I plasmid molecules are re- tained in the upper aqueous layer. One round of acid-phenol extraction is usually sufficient to obtain a preparation consisting of essentially 100% form I plasmid DNA. A schematic outline of the alkaline lysis/acid-phenol purifi- cation method is presented in Fig. 1. 2. Materials All stock solutions and buffers should be prepared using “nanopure” (double distilled and deionized) water and chemicals of the highest purity available (i.e., certified as molecular biology grade). In addition, and unless stated otherwise, buffers as well as all glass and plasticware should be autoclaved to inactivate contaminating deoxyribonucleases. Plasmid DNA Preparation Transformed bacterial strain 5 ml sterile LB-broth t antibiotic 0 Innoculate 250 ml to 1 L sterile LB-broth t antibiotic 500 ml (250 ml) Oak Ridge bottle(e) 30 ml Oak Ridge tube(s) 30 ml (15 ml1 Corex tube(s) 1.5 ml microcentrifuge tube a 0 a u a B 5 Grow overnight Grow until O.D.aee reaches 0.8 Add chloramphenicol (or spectinomycin) Continue growth for 20 hours Harvest/wash cells Lysis (PEB I, II, III) Precipitate plasmid DNA and bacterial RNA (twice) Bulk removal of bacterial RNA (LiCl-MOPS/heating) Ethanol precipitation of plasmid DNA (twice) RNAse A/T1 digestion Proteinase K digestion Phenol extzactions Rthanol precipitation of plasmid DNA Resuspend plasmid DNA in Tris-CDTA or sterile nanopure water Obtain pure form I plasmid DNA by acid-phenol extraction Fig. 1. Schematic outline of the alkaline $&/acid-phenol plasmid purification procedure. 6 Aubin, Weinfeld, and Paterson 2.1. lFiton &ysislCsCl Double Banding Method 1. Superbroth: Superbroth is prepared from stock solutions A and B. To prepare solution A, mix 120 g of tryptone, 240 g of yeast extract, and 50 mL glycerol in 9000 mL of water. Prepare solution B by mixing 125 g of %HPO, and 38 g of KHgO,in a total vol of 1 L. Sterilize each solution separately in the autoclave. Prepare 1 L of Superbroth by combining 900 mL of solution A and 100 mL of solution B. The final pH should be 7.2. 2. TE: 10 mMTris-HCl, pH ‘7.9; 1 mMEDTA, pH 8.0. 3. TES: 50 mM Tris-HCl, pH ‘7.5; 40 mM EDTA; 25% (w/v) sucrose. Sterilize by filtration (0.22 w). 4. 0.25MEDTA, pH 8.0. 5. Triton solution: Mix together 1 mL of 10% Triton X-100; 31.5 mL 0.25M EDTA, pH 8.0; 5 mL of 1 M Tris-HCl, pH ‘7.9; and 62.5 mL of water. Sterilize the solution by filtration (0.22 pm) and store at 4°C. 6. Ethidium bromide: 10 mg/mL in 10 mMTrisHC1, pH 7.5. This solu- tion does not require sterilization. ‘7. 5MNaCl. 8. HPLCgrade ethanol (95%). 9. Isopropanol. 2.2. Large-Scale Isolation of Plasmid DNA by Alkaline Lysis 1. LB (Luria-Bertani) broth: Dissolve 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl in 900 mL ofwater and adjust the pH to 7.5. Complete to 1 L with water and sterilize immediately in the autoclave. 2. NTE buffer: 10 mMTris-HCl, 100 mMNaC1, 1 mMEDTA (pH ‘7.5). 3. PEB I: 50 mM n-glucose, 25 mM Tris-HCl, 10 mM CDTA (pH 8.0). Sterilize the solution by filtration (0.22 pm) and store at 4OC. Do not autoclave. PEB I is stable indefinitely if stored under aseptic conditions. 4. PEB II: 0.2N NaOH, 1 .O% SDS. Clarify the solution by filtration (0.22 p) . Do not autoclave. Store at room temperature. PEB II is stable for at least 3 mo. 5. PEB III: 3Mpotassium acetate, 1.8Mformic acid. Clarify the solution by filtration (0.22 pm) and store at room temperature. Do not autoclave. PEB III is stable for at least 3 mo. 6. Acetate-MOPS: 100 mMsodium acetate; 50 mMMOPS, pH 8.0. Clarify the solution by filtration (0.22 pm) before sterilizing in the autoclave. 7. LiCL-MOPS: 5MLiCl; 50 mMMOPS, pH 8.0. This solution tends to be turbid even if high-purity LiCl is used. Clarify the solution by filtration (0.45 pm) before sterilizing in the autoclave. Plasmid DNA Preparation 7 8. RNAse buffer: 50 mMTris-HCl, 10 mMNaC1, 10 mMEDTA (pH 7.5). 9. RNAse A (DNAse-free): Dissolve lyophylized RNase A to a final concen- tration of 1 mg/mL in sterile 5 mM Tris-HCl, pH ‘7.5. Hold the preparation at 80°C for 15 min to inactivate traces of contaminating deoxyribonucleases, and allow to cool at room temperature for 20 min. Dispense 1 00-PL vol in sterile microcentrifuge tubes and store at -2OOC. Individual tubes may be thawed and refrozen repeatedly without signifi- cant loss of enzyme activity. 10. RNAse Tl : Concentrated RNase Tl (125,000 U/mL) is diluted to a final concentration of 500 U/mL in sterile 5 mMTris-HCl, pH ‘7.5, and stored at 4OC. 11. 10% SDS: Dissolve 10 g of molecular biology grade sodium dodecyl sulfate (SDS) in 100 mL of autoclaved nanopure water. Clarify the so- lution by filtration (0.22 pm) and store at room temperature. Do not autoclave. 12. Proteinase K: Dissolve lyophylized proteinase K to a final concentration of 20 mg/mL in autoclaved nanopure water. Dispense lOO-pL vol in sterile microcentrifuge tubes and store at -2OOC. Individual tubes may be thawed and refrozen repeatedly without significant loss of potency. 13. T&-buffered phenol (pH 8.0): The use ofhigh-purity (redistilled) phenol is critical. Begin by melting crystallized phenol in a 65OC water bath. Add an equalvol of lMTris-HCl, pH 8.0/0.2% ~mercaptoethanol. Shake vigorously to create an emulsion, and allow the phases to separate by gravity. Remove the upper aqueous layer by aspiration and equilibrate the phenol twice with an equal vol of O.lM Tris-HCl, pH 8.0/0.2% pmercaptoethanol. Store the buffered phenol in the dark at 4OC under a film of O.lMTris-HCI, pH 8,0/0.2% pmercaptoethanol. The prepa- ration remains stable for at least 3 mo under these conditions. CAUTION: Phenol is a strong oxidant and can cause severe burns. Always wear gloves and exercise due care in handling. Phenol is also light-sensitive. Always prepare and store in an amber glass bottle. 14. Phenol:chloroform (1:l): Add 1 vol of Tris-buffered phenol to an equal vol of chloroform:isoamyl alcohol (24:l) (see below) in an amber glass bottle. Store in the dark at 4OC under a film of O.lMTrisHCl, pH 8.0/ 0.2% ~mercaptoethanol. 15. Chloroform:isoamyl alcohol (24:l): Mix 240 mL of chloroform and 10 mL of isoamyl alcohol in a glass amber bottle. Store in the dark at 4OC. 16. 2.5Msodium acetate, pH 5.5. 17. Tris-CDTA: 10 mMTris-HCl; 1 mMCDTA, pH 8.0. 18. HPLC-grade ethanol (95%). 8 Aubin, Weinfeld, and Paterson 19, Lyzozyme (lyophylized) . 20. Chloramphenicol: Prepare a 34mg/mL stock solution using 95% eth- anol just prior to use. 21. Spectinomycin: Spectinomycin is usually present at 66% potency. Prepare as required at a stock concentration of 30 mg/mL in nanopure water. Sterilize by filtration (0.22 pm) before use. 2.3. Purification of Form I Plasmid DNA by Acid-Phenol Extraction 1. 50 mMsodium acetate/75 mMNaC1, pH 4.0: To 400 mL of autoclaved nanopure water, add 10 mL of 2.5Msodium acetate, pH 5.5, and ‘7.5 mL of a 5MNaCl stock solution. Adjust the final pH exactly to 4.0 with gla- cial acetic acid, and complete the vol to 500 mL. Sterilize the solution by filtration (0.22 pm) 0nZy and store at room temperature. 2. 50 mM sodium acetate, pH 4.0: To 400 mL of autoclaved nanopure water, add 10 mL of 2.5M sodium acetate, pH 5.5. Adjust the final pH to 4.0 with glacial acetic acid and complete the vol to 500 mL. Sterilize the solution by filtration (0.22 pm) only and store at room temperature. 3. Acidified phenol: Melt high-purity (redistilled) phenol in a 65OC water bath. Add an equal vol of 50 mM sodium acetate, pH 4.0, and shake vigorously to create an emulsion. Allow the phases to separate by gravity and remove the upper aqueous layer by aspiration. Repeat this proce- dure twice. Store acidified phenol in the dark at 4OC under a film of 50 mM sodium acetate (pH 4.0). This product has a short shelf-life and should be prepared either as required or on a biweekly basis. 3. Methods 3.1. Isolation of Form I Plasmid DNA by lkiton Llysis Followed by CsCl Banding 1. Initiate a 5-mL culture from a single colony (or frozen stock) of bacteria containing the plasmid at 37°C overnight. Use the entire culture to seed 800 mL of Superbroth, and incubate for 36 h at 37°C with vigorous shaking. 2. Collect the bacteria by centrifigation at 5000gfor 10 min (4OC), using a Sorvall GS-3 rotor (or its equivalent; DuPont). 3, Resuspend the bacteria in 100 mL of TE. Centrifuge again to pellet the material. The sample may be stored frozen (-20°C) at this stage. 4. Resuspend the pellet thoroughly in 9 mL of ice-cold TES. 5. Add 0.9 mL of 10 mg/mL lysozyme (freshly prepared in TEYS) and hold on ice for 5 min. Plasmid DNA Preparation 9 6. Add 3.7 mL of 0,25MEDTA, pH 8.0, mix thoroughly, and hold on ice for another 5 min. ‘7. Add 14.5 mL of ice-cold Triton lysis solution, mix thoroughly, and hold on ice for 10 min. 8. Remove the debris by centrifugation (25,000 rpm in a Beckman SW2’7 rotor or its equivalent) for 30 min at 4OC. 9. Decant the supernatant into a 50-mL polypropylene tube and adjust the weight of the liquid to 30.17 g with TE. Add 28.14 g of solid CsCl and mix thoroughly until completely dissolved. Add 4.5 mL of ethidium bromide solution and mix by inversion. CAUTION: Ethidium bromide is a very strong mutagen. Always wear gloves while handling it. 10. Transfer the mixture to a Beckman polyallomer Quick Seal@ ultracen- trifuge tube, and seal the vessel according to the supplier’s instructions. A single preparation can be accommodated by the Beckman VTi 50 or Ti 60 rotor. The VTi 50 rotor should be spun for at least 18 h at 45,000 rpm (20°C)) whereas the Ti 60 unit will require a run time of at least 60 h at 35,000 rpm (2OOC). NOTE: The new generation of Beckman rotors and benchtop ultracentrifuges allows for smaller plasmid preparations to be banded in much shorter time periods. 11. Following centrifugation, view the tubes under medium-wave (302-nm) W light. CAUTION: Ultraviolet light is both mutagenic and carcino- genic. Wear adequate protection (i.e., use a W-protective face shield [eye goggles will not protect the rest of your face or neck], ensure that your lab-coat sleeves cover your forearms, and wear latex gloves), Se- cure the tube and insert a 19-gage syringe needle at the top to create an air inlet. Puncture the side of the tube at a point just below the lower- most band (form I plasmid; the upperband consists of open circular and linear molecules) using a 20-mL hypodermic syringe equipped with a 19gage needle. Collect the banded material in a total vol of 4-5 mL. 12. Either reband the plasmid sample (seestep 13) or remove the ethidium bromide with isopropanol saturated with CsCl solution at the concentra- tion used for banding. Dialyse extensively against a large vol of TE to remove the CsCl. Add l/10 vol of 5MNaCl and 2 vol of ethanol. Allow the plasmid to precipitate overnight at -20°C, and collect the mate- rial by centrifugation at 10,OOOg for 15 min at 4OC. Drain away the ethanol carefully and air-dry the pellet to remove all traces of ethanol. 13. It is often necessary to reband the plasmid preparation. To do this, prepare a 1.08 g/mL CsCl solution in TE. Add 0.1’7 mL of ethidium bromide solution for each mL of TE used. Add the DNA sample obtained in step 12 to the solution, mix thoroughly, centrifuge, and process as in steps 1 O-21. 10 Aubin, Weinfeld, and Paterson 3.2. Large-Scale Isolation of Plasmid DNA by Alkaline L&J 1. Revive a stock of plasmid-bearing bacteria in 5 mL of LB broth contain- ing the antibiotic(s) required for plasmid selection. Incubate overnight at 37°C with shaking. The next morning, transfer 2.5 mL of the satu- rated culture to two 2-L Erlenmeyer flasks (each containing 500 mL of sterile antibiotic/LB broth), and grow the bacteria at 37°C with vigorous shaking (250 x-pm) until the OD, reaches 0.8. Add 2.5 mL of the 34 mg/mL chloramphenicol stock (final concentration, 170 pg/mL) to each flask and allow plasmid amplification to proceed over 20 h at 37°C with shaking. Plasmids containing a transcriptionally active chlorampheni- co1 acetyl transferase cassette can be amplified using 150 pg/mL spectinomycin. 2. Harvest the bacteria by centrifugation at 500gfor 8 min (4OC) in 500-mL Oak Ridge bottles using a Sorvall GS3 rotor or its equivalent. Discard the supernatant into a receptacle containing germicidal detergent or liquid bleach. 3. Resuspend each bacterial pellet in 50 mL of ice-cold NTE buffer. Pool the material in a single 250-mL Oak Ridge bottle and recover the washed cells by centrifugation at 5000gfor 8 min at 4°C using a Sorvall GSA r-o- tor or its equivalent. Discard the supernatant as in step 2. 4. Using a sterile l-mL plastic pipet as a stirring rod, gently dissolve the pellet in 1 mL of icecold PEB I buffer. A homogeneous slurry should be obtained. Add 9 mL of icecold PEB I buffer supplemented with 10 mg of lysozyme, and mix thoroughly with the plastic pipet. Allow cell lysis to proceed for 30 min in ice water (seeNote 2). 5. Add 20 mL of room-temperature PEB II and stir the mixture thoroughly but gently with the plastic pipet. The lysate should become very viscous and translucent. Hold in ice water for 15 min and stir occasionally. 6. Add 15 mL of room-temperature PEB III and stir the mixture vigorously with the plastic pipet for several min until a coarse white precipitate forms and the viscosity disappears. Hold in ice water for 30 min and collect the debris by centrifugation at 10,OOOgfor 10 min (4OC). 7. Transfer the supernatant to a new 250-mL Oak Ridge bottle and add 2 vol (90 mL) of chilled (-20°C) ethanol. Mix well and precipitate the nucleic acids at -70°C for 30 min. Collect the material by centrifugation at 10,OOOg for 10 min (4OC). 8. Using a sterile pipet, gently dissolve the pellet in 5.0-7.5 mL of acetate-MOPS buffer. Transfer the solution to a sterile 30-n& Oak Ridge or Corex@ tube and precipitate the nucleic acids at -70°C for 30 min. Collect the material by Plasmid DNA Preparation 11 centrifugation at 10,OOOg for 10 min (4OC) using a Sorvall HB4 or SS34 rotor. 9. Decant the ethanol carefully and resuspend the pellet in W-7.5 mL of autoclaved nanopure water. Measure the vol of the sample and add an equal vul of LiCl-MOPS buffer. Mix well and place in ice water for 15 min. Centrifuge at 10,OOOg for 10 min (4OC). This step will precipitate the bulk of the coextracted RNA and leave the plasmid DNA (with re- sidual low-mol-wt RNA species) in solution. 10. Using a sterile pipet, transfer the supernatant to a new Oak Ridge or Corex@ tube. Hold at 60°C for 10 min, and collect any residual precipi- tate by centrifugation as in step 9 (see Note 3). 11. Divide the supernatant between two 30-mL Corex@ tubes and add 2 vol of chilled ethanol. Hold at -70°C for about 1 h and recover the nucleic acids by centrifugation at 10,OOOgfor 10 min (4OC). 12. Resuspend each pellet in 5.0-7.5 mL of acetate-MOPS buffer and com- bine the material in one of the Corex@ tubes. Precipitate the sample with 2 vol of chilled ethanol as outlined in step 8. Repeat the precipitation once. Decant the ethanol carefully and air-dry the pellet for 10-15 min. 13. Dissolve the pellet in 5.0-7.5 mL of RNase buffer. Add 10 pL/mL RNase A stock (final concentration 10 pg/mL) and 10 pL/mL RNase Tl stock (final concentration 5 U/mL) . Incubate at 37°C for 90 min. 14. Add 50 pL/mL 10% SDS stock (final concentration 0.5%) and 2.5 pL/ mL proteinase K stock (final concentration 50 pg/mL). Incubate for 2 h at 37°C. 15. Extract the aqueous mixture as follows: once with Tris-buffered phenol, twice with phenol:chloroform (l:l), and once with chloroform:isoamyl alcohol (24:l). Disposable polypropylene culture tubes (sterile; 17 x 100 mm) are very practical for this purpose. For each extraction step, add an equal vol of the organic solvent, create an emulsion by shaking (do not use a mechanical vortex), and separate the phases by centrifu- gation at 5000g for 5 min (25°C). A Sorvall HB4 rotor allows sharp phase separation. Transfer the upper aqueous phase to a new tube fol- lowing each extraction step. 16. To the final aqueous phase, add l/10 vol of 2.5Msodium acetate, pH 5.5, and 2 vol of chilled ethanol. Precipitate the plasmid DNA for 20 min at -70°C and centrifuge as described in step 8. 17. Clean the plasmid DNA twice by acetate-MOPS/ethanol precipitation as described in step 12. Dry the DNA pellet briefly under vacuum and dissolve in 1 mL of sterile Tris-CDTA buffer. Avoid using a mechanical vortex to assist solubilization. Determine the concentration of the plas- mid DNA stock by taking the absorbance of a l/20 dilution at 260 nm 12 A&in, Weinfeld, and Paterson and assuming that 1 .O OD, unit is equivalent to a concentration of 50 pg DNA/mL. For example, mix 25 / tL of the DNA preparation in 475 PL of Tris-CDTA. Using a 0.5 mL quartz cuvet (glass cuvets absorb sign&an tly in the W spectrum), “zero” the spectrophotometer using Tris-CDTA. Take the OD reading of the DNA and calculate its concen- tration as follows: (reading at 260 nm) x 20 (l) X 50 W= [DNA] in pg/mL where (I) is the dilution factor and (*) is the conversion factor. The sample should also be scannedwithin the 200-to 300-nm range in order to obtain a rough measure of the purity of the preparation. The scan should show two peaks, the first around 205 nm (attributed to the Tris-CDTA) and the other at 260 nm (contributed by DNA), A high-purity DNA preparation should show no evidence of a shoulder beyond 260 nm, and the 260 nm/280 nm ratio should be >1.8. If care is taken throughout the procedure to pipet gently and mini- mize the use of a mechanical vortex, form I plasmid DNA is routinely obtained at >90% yield for plasmids of 15 kbp or less, as verified by aga- rose gel electrophoresis. 3.3. Purification of Form I Plasmid DNA by Acidified-Phenol Extraction 1, Transfer the DNA stock to a sterile 1.5-mL microcentrifuge tube. Add l/10 vol2.5Msodium acetate, pH 5.5, mix well, and add 2 vol of chilled ethanol. Precipitate the DNA at -‘70°C for 30 min and recover by cen- trifugation at 10,OOOg for 10 min (4OC). Air-dry the pellet for 15 min at room temperature. 2. Gently redissolve the sample to a final concentration not exceeding250 yg/mL in 50 mM sodium acetate/‘75 mMNaC1, pH 4.0. A total vol of 400-500 I,~L is practical (see Notes 4 and 5). 3. Add an equal vol of acidified phenol and shake vigorously by hand (DO NOT VORTEX) for 2-3 min. Separate the phases by centrifuga- tion at 10,OOOgfor 60 s at room temperature. 4. Transfer the upper aqueous layer to a sterile 1.5-mL microcentrifuge tube and precipitate the plasmid DNA as outlined in Step 1. Redissolve the pellet in 250 PL of acetate-MOPS buffer and precipitate the DNA with 2 vol of chilled ethanol. Repeat this step once. Resuspend the DNA sample in 0.5 mL of TrisCDTA. Determine the concentration and verify the integrity of the preparation by spectrophotometry and agarose gel electrophoresis, respectively. One round of acid-phenol extraction is generally sufficient to achieve a high degree (i.e., > 98% form I) of plas- mid purification. [...]... Stokoe, N., and Siminovitch, L (1980) Parameters governing the transfer of the genes for the thymidine kinase and dihydrofolate reductase into mouse cells using metephase chromosomes or DNA Somatic Cell Genet 6, 333-348 15 Faber, F E and Eberle, R (19’76) Effects of cytochalasin and alkaloid drugs on the biological expression of herpes simplex virus type 2 DNA Exp Cell Res 103, 15-22 16 Luthman, H and Magnusson,... Es&erichia coli gene coding for xanthine-guanine phosphoribosyltransferase Proc Null Acad Sci USA 78,2072-2076 20 Kaster, K R, Burgett, S G., Nagaraja, R, and Ingolia, T D (1983) Anal@ ofa bacterial hygromycin B resistance gene by transcriptional and translational fusion and by DNA sequencing Nuckk Acids Res 11,6895-6911 hAPTER 3 Xkansfection of the ChloramphenicolAcetyltransferase Gene into Eukaryotic... to the chloramphenicol acetyl transferase (CAT) gene This type of gene transfer is often referred to as a transient expression system Production of the CAT enzyme peaks at around 40-48 h and thereafter the level falls as the plasmid DNA is diluted out in a growing population of cells The general strategy of subcloning putative regulatory regions followed by transfection and quantification of CAT activity... population, and dimethyl sulfoxide (DMSO)is then employed to permeabilize the DNAcoated cells The method is simple to perform and has proven very effective, producing stable transfection frequencies on the order of O.Ol-O.l% with only From: Methods in Molecular Biology, Vol 7: Gene Transfer and Expression Protocols Edited by: E J Murray 0 1991 The Humana Press Inc., Clifton, NJ 35 36 Aubin, Weinfeld, and Paterson... Trypsinize and split cells at an appropriate ratio (>l :lO) Refeed and incubate for another 24 h under regular growth conditions before selection (see Notes 4 and 5) For assaying transient expression of transfected DNA, incubate the cells for 48 h without splitting 4 Notes 1 The in vitro precipitation method is relatively insensitive to the amount of DNA and the culture conditions used, and generally... Greene, M I., and Weinberg, R A (1984) The nue oncogene: An e&B-related gene encoding a 185,000-M, tumour antigen Nature 321,513-616 7 Shih, C and Weinberg, R A (1982) Isolation of a transforming sequence from a human bladder carcinoma cell line Cell 29, 161-169 8 Shimizu, K., Coldfarb, M., Perucho, M., and Wigler, M (1983) Isolation and preliminary characterization of the transforming gene of a human... Methods in Molecular Biology, Vol 7: Gene Transfer and Expression Protocols Edited by: E J Murray 01991 The Humana Press Inc., Clifton, NJ 23 24 Lake and Owen Fig 1 Flow diagram showing the key steps in transient transfection analysis (1) Putative regulatory regions are excised from genomic DNA by restriction enzyme digestion Fragments are (2) subcloned into the CAT vector and (3) transfected into eukaryotic... constructions pBLCAT2 and pBLCAT3 (2) because both have multiple unique restriction sites, both 5’ and 3’ of the CAT gene (Fig 2) In general, plasmid vectors must contain an origin of replication and an antibiotic resistance marker These sequences allow the amplification and selection of the plasmid in a bacterial host Eukaryotic control elements must also be present to achieve efficient expression in transfected... advisable to confirm and extend an analysis of enhancer specificity, using the homologous promoter One recent study in transgenic mice underlined this problem clearly A globin gene under the control of its own promoter and the CD2 enhancer was expressed in both T-cells and erythroblasts (6) In conclusion, it seems likely that tissue specific gene expression can result from both promoter and enhancer elements... 56,979-986 7 Maniatis, T., Goodbourn, S., and Fisher, J A (1987) Regulation of inducible and tissue specific gene expression Science 236,1237-1245 8 Maniatis, T., Fritsch, E F., and Sambrook, J (1982) Molecular Cloning (A Labmzto~ Manual) (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York) 9 Sussman, D J, and Milman, G (1984) Short term, high efficiency expression of transfected DNA Mol Cell . Methods in Molecular Biology, Vol. 7: Gene Transfer and Expression Protocols Edited by: E. J. Murray 0 1991 The Humana Press Inc., Clifton, NJ 3 Aubin, Weinfeld, and Paterson equipment. As a result,. Methods in Molecular Biology, Vol. 7: Gene Transfer and Expression Protocols Edited by: E J Murray 0 1991 The Humana Press Inc., Clifton, NJ 15 16 Okayama and Chen The calcium phosphate method. specialized and versatile eucaryotic cloning /expression vectors, investigators have been given powerful tools to expedite the elucidation of mechanisms governing gene expression, and to facilitate