Preparation of Wild Type or Recombinant SV40 Stocks

3.1.1. SV40 as a Vector for Cellular Promoters

1. Clone the cellular promoter with a reporter gene, if desired, (e.g., ß-globin, chloramphenicol acetyltransferase) in place of the SV40 early genes in an SV40 recombinant bacterial plasmid. A convenient plasmid for insertion of exogenous DNA is pSVSX-T (see Note 8). The parent of this plasmid, pSVSX, is a SV40/ pBR322 recombinant plasmid, fused at the respective £coRI restriction sites, containing a single XhoI site at position 2666 in the SV40 genome. Subsequent deletion of SV40 sequences 2770 (BclI) to 5027 (£coNI) removed the early coding sequences, yielding pSVSX-T (Fig. 1A). Cloning of exogenous sequences into the unique XhoI site results in plasmids such as pSVSpS2 (30), which contains the human pS2 promoter from -1100 to +10 driving rabbit ß-globin coding sequences (Fig. 1B). For optimal packaging of recombinant viral DNA into an SV40 virion, the length of the recombinant genome should be no larger than 5-9% greater than the length of a wild-type genome (5243 bp), with more-efficient packaging the closer it is to the wild-type length (37). In the case of pSVSX-T, this translates into a DNA insert of 2260 bp to result in a wild-type length genome and DNA inserts not exceeding 2700 bp (including both the cellular regulatory sequences and any reporter gene) for the highest efficiency of packaging. It is preferable to insert the promoter (and reporter gene) such that the promoter is positioned in an opposite orientation to that of the endogenous t/T genes being replaced in order to distance the inserted transcriptional elements as far from the SV40 enhancer sequences as feasible (1000-2000 bp).

2. Precisely remove the bacterial plasmid sequences from the recombinant viral genome by digestion with an appropriate restriction enzyme (limited digestion with EcoRI, in the case of the construct in Fig. 1B). Recircularize the DNA with ligase, using sterile ligation buffer (yielding the recombinant viral genome, as in Fig. 1B). The DNA concentration should be low to promote circularization over concatamerization (e.g., 0.75 ng DNA/pL ligation mixture). Heat inactivate the ligase for 10 min at 65°C. Verify by gel electrophoresis that the recircularization was successful.

3. Prepare a stock of the desired recombinant virus using a limiting dilution trans-fection protocol (see Note 9), as follows. Prepare tenfold serial dilutions of the ligated DNA in sterile 10 mM Tris-HCl, pH 7.8, 10 mM EDTA. To 100 pL of each dilution of DNA, add 80 pL of 5 mg/mL DEAE-dextran (sterile and freshly prepared) and 220 pL of 2X DME + 10% fetal calf serum. Use dilutions ranging

B SV40

SV40 late genes

Fig. 1. Diagrams of plasmids instrumental in constructing recombinant SV40 viruses. (A) Diagram of pSVSX-T, a fusion of pBR322 and SV40 DNA sequences through their respective £coRI sites. The SV40 late genes are interrupted by pBR322 sequences. The positions of the SV40 late polyadenylation site (polyA), the SV40 origin of DNA replication (ori), and the unique XhoI site, for insertion of exogenous, cellular promoters, and reporter genes, are shown. (B) Diagram of pSVSpS2, a representative recombinant SV40 genome fused to pBR322 sequences. pSVSpS2 was derived from pSVSX-T by inserting human pS2 promoter sequences (-1100 to +10) and a 6-globin reporter cDNA into the XhoI site of pSVSX-T. In addition to landmarks shown in (A), the pS2 promoter sequences are shown by dark stippling and the 6-globin sequences by light stippling.

SV40 late genes

Fig. 1. Diagrams of plasmids instrumental in constructing recombinant SV40 viruses. (A) Diagram of pSVSX-T, a fusion of pBR322 and SV40 DNA sequences through their respective £coRI sites. The SV40 late genes are interrupted by pBR322 sequences. The positions of the SV40 late polyadenylation site (polyA), the SV40 origin of DNA replication (ori), and the unique XhoI site, for insertion of exogenous, cellular promoters, and reporter genes, are shown. (B) Diagram of pSVSpS2, a representative recombinant SV40 genome fused to pBR322 sequences. pSVSpS2 was derived from pSVSX-T by inserting human pS2 promoter sequences (-1100 to +10) and a 6-globin reporter cDNA into the XhoI site of pSVSX-T. In addition to landmarks shown in (A), the pS2 promoter sequences are shown by dark stippling and the 6-globin sequences by light stippling.

from 7.5 ng DNA to 7.5 x 10-6 ng DNA/100 pL. Trypsinize CMT cells, preparing a single-cell suspension of cells at 5 x 105 cells/mL in DME + 5 % fetal calf serum. Add 400 pL DNA to each 400-pL aliquot of cells, using sterile microcentrifuge tubes. Invert tubes; incubate in shaking water bath at 37°C for 35 min. Centrifuge 5 min at 14,700g at 4°C in J6 centrifuge, to pellet cells. Wash each tube of cells 2X with 0.5 mL DME, removing supernatant each time with sterile, plugged Pasteur pipet. Resuspend each tube of cells in 10 mL DME, 5% fetal calf serum, 100 yM ZnCl2, 10 yM CdSO4 plus antibiotics. Pipet 1-mL cell suspension into each well of a 24-microwell plate. Use a set of 10 wells/dilution of ligated DNA. Include a mock-transfected, control set of cells for comparison of the morphology of the cells at subsequent times.

4. Monitor the state of the cells daily during the incubation of the microwell plates of cells for the subsequent 2-4 wk. After an initial 4 d of incubation, change the media, and as necessary thereafter, add more medium to the wells to prevent evaporation and pH changes. When cells demonstrate cytopathic effect (CPE) by rounding up and dying, collect the media from each well. The set(s) of cells to analyze further are those transfected by a given dilution of DNA, resulting in the cells in some, but not all, of the wells demonstrating CPE. These cells received the least amount of DNA necessary to achieve viral production and are therefore least likely to have undergone recombination during DNA transfection and most likely to contain unrearranged viral genomes. If the transfection method is capable of delivering one molecule of DNA per cell, one can be 95% confident that CPE in a well resulted from a single DNA molecule entering the cells when only 10% of the cultures receiving this dilution of DNA develop infection.

5. Harvest the virus by collecting the media and cells after pipetting up and down to detach any remaining cells attached to the plate. Sonicate the cellular suspension in a cup sonicator for 1 min to release any attached virus into the supernatant, and sediment the cellular debris from the sonicated suspension by centrifugation at low speed (see Note 10). To store the viral stock, quick freeze the supernatant in a dry ice:ethanol bath and store in a -70°C freezer. Such SV40 viral stocks are stable for several years with only minimal loss of titer.

6. Analyze the genomic DNA in the virus stocks by incubating 10 yL viral stock, 2 yL 10X dissociation buffer, and 8 yL H2O at room temperature for 30 min. Add 20 yL of 2X proteinase K and incubate an additional 30 min at 37°C. Extract with phenol: chloroform:isoamyl alcohol, then with chloroform, and precipitate with ethanol.

7. Purify the viral DNA, using anion exchange chromatography (e.g., Qiagen silica gel particles or glass milk), by the manufacturer's or common procedures (see Note 11).

8. Resuspend the DNA, digest with the appropriate restriction enzymes, and analyze by Southern blotting to determine which virus stock contains the desired, unrearranged, and complete viral genome sequence. To verify length of critical DNA fragments, compare with digestion of the input plasmid DNA. Pick two or more viral stocks to amplify further.

9. Amplify the viral stock, always saving some of the original isolate in case it is needed. To infect, remove medium from 40 15-cm-diameter plates of 70-80% confluent CMT cells and rinse the cells with DME. Pipet dropwise 100 yL of the viral stock uniformly across each plate, rocking briefly back and forth. Add 1 ml DME to prevent evaporation that leads to cell death and adsorb the virus for 1-2 h at 37°C in the CO2 incubator. Add 10 mL DME, 2% fetal calf serum, 100 yM ZnCl2, 10 yM CdSO4, and antibiotics to the plates (see Note 12), and incubate 2-4 wk, until the cells demonstrate uniform CPE. Always maintain mock-infected plates of cells in order to compare cell morphology. Supplement (or remove and replace) the media on the plates every 3-6 d (see Note 13). Take care to change media on the control plates separately from the infected plates to avoid cross-contamination with virus.

10. Sterilely harvest the virus stock:

a. Scrape the cells and combine with the media, which contains a large amount of cellular debris, into a large centrifuge bottle.

b. Sediment the cells and debris by centrifugation at 5300g in a J6 centrifuge for 5 min at 4°C.

c. Sonicate the cellular pellet, resuspended in a small volume of media, in a cup sonicator for 1 min, to release any attached virus into the supernatant.

d. Sediment the cellular debris from the sonicated suspension, as above.

e. Combine the supernatant from the sonicated pellet with the supernatant media from the first centrifugation step.

f. Aliquot the virus stock, freeze the aliquots in a dry ice:ethanol bath, and store in a -70°C freezer (see Note 10).

11. Titer the recombinant SV40 stock by the end-point dilution method (38) (see Note 14). Prepare tenfold serial dilutions of the virus stock in DME in a final vol of 200 yL, in sterile microcentrifuge tubes. Make certain to change pipettes for each subsequent dilution. Dilutions should range from 10-1-10-8 (see Note 15). In addition, prepare intermediate dilutions of 5 x 10-6, 5 x 10-7, and 5 x 10-8. For each dilution, ranging from 10-3-10-8, use 100 yL of the virus per well to infect at least four wells of a 24-microwell plate that are 70-80% confluent with CMT cells. Include mock-infected control wells, using 100 yL DME. Following adsorption of the virus at 37°C for 1-2 h, remove the medium and replace with fresh DME plus 2% fetal calf serum, 100 yM ZnCl2, 10 yM CdSO4, and antibiotics. Monitor the state of the cells daily for CPE during the incubation of the microwell plates of cells for the subsequent 2-4 wk. After an initial 4 d of incubation, and as necessary thereafter, add more medium to the wells to prevent evaporation and pH changes. The final percentage of wells that develop CPE at the varying dilutions of virus can be used to determine the 50% tissue culture infectious dose (TCID50), which can be converted to plaque-forming units (pfu) by the following equation: TCID50/mL x 0.69 = pfu/mL. An approximate titer of the virus, sufficient for all subsequent procedures in the isolation of MCs, is obtainable by extrapolating from the percentage of wells with CPE at each dilution of virus to what viral concentration would result in a 50% infection rate. A more precise determination of the TCID50 (at least 10 wells of cells required per virus dilution), using the methodology of Reed and Muench, is fully described in many laboratory manuals regarding baculovirus (38).

12. To verify that the amplified viral stock is not contaminated with variants, which might ultimately overgrow the population upon propagation (see Note 16), recombinant viral DNA from the expanded stock must be prepared and analyzed. Infect 20-30 15-cm-diameter tissue culture plates of CMT cells, at 70-80% confluence, with virus, ideally at a multiplicity of infection (moi) of between 5-10 pfu/cell to ensure infection of 100% of the cells. At lower moi's, fewer cells would be infected resulting in lower yields of DNA (e.g., at a moi of 1 pfu/cell, 63% of the cells are infected, as determined by the Poisson distribution curve). As described above, remove media from the cells and rinse with DME prior to addition of virus and adsorption for 1-2 h in a CO2 atmosphere at 37°C. Then, add DME plus 2% fetal calf serum, 100 |M ZnCl2, 10 |M CdSO4, and antibiotics to a total volume per plate of 15 mL. Harvest the viral DNA between 48 and 72 h postinfection (see Note 17):

a. Remove the media.

b. Wash the cells with PBS.

c. Add 2 mL Hirt extraction buffer per plate, rocking to cover the entire plate.

d. Incubate 15 min at room temperature.

f. Scrape with rubber policeman into centrifuge tubes (30-mL Corex tubes).

g. Incubate on ice overnight (or for at least 2 h).

h. Centrifuge at 4°C for 60 min at 21,000g in an SS-34 rotor in a high speed centrifuge.

i. Collect the supernatant (the pellets should be firm).

j. Add 0.815 g CsCl/mL of supernatant; then add 0.1 mL of 10 mg/mL ethidium bromide/mL of supernatant. k. Centrifuge in a NVT90 rotor at 410,000g for 4 h (long enough to pellet contaminating RNA).

l. Remove the lower DNA band, and repeat the CsCl-ethidium bromide cen-trifugation step if transcription-grade DNA is desired. m. Extract the DNA solution three times with isopropanol to remove the ethidium bromide.

n. Dialyze the DNA in two changes of 1 L of 10 mM Tris-HCl, pH 8.3, 1 mM EDTA.

The viral DNA can be analyzed by restriction enzyme digestion, Southern blotting, and sequencing, to ensure that the sequence structure is as desired. This DNA also provides an essential reagent for comparing the properties of chroma-tin and DNA templates when transcribing the corresponding MCs in vitro (see Subheading 3.4.).

3.1.2. Propagation of SV40

1. Wild type SV40 virus is propagated in CV-1 cells. Our CV-1 cells are propagated in DME + 10% calf serum in a 5% CO2 atmosphere at 37°C. Infected cells are maintained in DME + 2% calf serum. In order to propagate viral stocks, infect CV-1 cells with SV40 virus at a moi of 0.001 (see Note 16). Proceed with the infection and harvest as described in steps 9 and 10 in Subheading 3.1.1., growing the CV-1 cells in the presence of calf serum, and in the absence of zinc and cadmium divalent cations.

2. Wild type SV40 is most directly titered by plaque assay, using CV-1 cells. Prepare tenfold serial dilutions of the virus stock, from 10-1-10-8, into DME. Make certain to change pipettes for each subsequent dilution. Rinse ten subconfluent, 60-mm-diameter plates of CV-1 cells with DME. Add 0.1 mL of each viral dilution dropwise over the entire surface of the plate, in duplicate. Use the 10-5, 10-6, 10-7, and 10-8 viral dilutions, as well as duplicate control plates that are mock-infected with just DME. Allow viral adsorption by incubating in the 37°C CO2 incubator for 1-2 h. During that time, prepare a 1.8% bactoagar solution by heating the agar in water in a microwave oven. Incubate in a 48°C water bath, allowing the agar sufficient time to cool to 48°C. Just prior to overlaying the infected plates of cells, combine equal volumes of 2X DME + 4% calf serum + antibiotics with 1.8% bactoagar. Immediately add 5 mL of the mixture onto each plate of cells, pipetting down the inside wall of the plate so as to avoid any disturbance in the lawn of cells. Permit the agar to set for approx 30 min before moving the plates back into the 37°C incubator. Monitor the plates of cells for the next 1-2 wk for the appearance of plaques. Refeed the plates at least once, by overlaying an additional 2-3 mL bactoagar:DME:2% calf serum:antibiotics over the previous agar. To facilitate final quantitation of the numbers of plaques, prepare the bactoagar:DME:serum solution as before, but including neutral red to a final concentration of approximately 0.01%. Overlay each plate with 2 mL of this mixture. Wrap the set of plates in aluminum foil to exclude light. One to four days later, count the plaques (clear circles on a red background) on plates where plaques are distinct and well separated. This will provide the titer of the original virus stock, as expressed in pfu per milliliter of stock.

3. If the virus stock has not been carefully propagated, and/or analysis of its DNA reveals the presence of multiple viral genomes in the stock, the virus requires purification. This can be accomplished most readily by plaque purification (see Note 18). Obtain plaques from the viral stock as described (see Subheading 3.1.2., step 2). On a plate on which plaques are well separated, pick plugs containing plaques by puncturing through the entire agar overlay with a sterile Pasteur pipet. Combine each plug with 1 ml DME plus antibiotics. Harvest by three cycles of freezing in dry ice:ethanol bath and thawing in a 37°C water bath. To store, refreeze in dry ice:ethanol bath and keep in -70°C freezer. To ensure purity of the virus stock, repeat the plaque purification procedure once a plaque isolate has been identified that contains the wild type genomic sequence. Then, amplify the viral stock as described (see Subheading 3.1.2., step 1).

4. As controls for structural and transcriptional analyses with SV40 MCs, prepare and purify SV40 genomic DNA by the Hirt extraction protocol. This protocol is described (see Subheading 3.1.1., step 12) for recombinant viruses requiring growth in CMT cells. Perform parallel procedures, except using CV-1 cells and media containing calf serum, without the ZnCl2 or CdSO4. Quantitate the amount of viral DNA by obtaining the UV absorbance of a dilution of the DNA.

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