Transient Replication and Plasmid Maintenance Assays in Mammalian Cells

The replication and maintenance of plasmids containing OriP (negative control) or containing OriP and expressing the EBNA-1 gene (positive control) is measured in human cells as described below in the transient replication and plasmid maintenance assays. The plasmid maintenance assay measures a combination of DNA replication and segregation efficiency and reflects segregation ability when DNA replication occurs normally. The main differences in the transient replication and plasmid maintenance assays are in the amount of DNA transfected, the length of time that the plasmids are left in the cells, and the fact that the plasmid maintenance assay involves selecting for the plasmid. The steps describe (1) construction of the plasmids used in the assays, (2) transfection of a human cell line with the plasmids, (3) growth of the transfected cells, (4) extraction of the plasmids from the cells, and (5) Southern blot analysis of the extracted plasmids to determine whether they replicate and/or can be maintained in the cell line used.

3.1.1. Plasmids

Subheadings 3.1.1.1.-3.1.1.3. describe the backbone plasmid used to construct the plasmids used in the transient replication and maintenance assays, the DNA fragments containing OriP or the EBNA-1 gene, and the strategy used to clone these DNA fragments into the backbone plasmids.

pcDNA3 (Fig. 1) is a 5.4-kb vector that contains a bacterial origin of replication (colEl) and the ampicillin resistance marker, allowing its propagation in bacteria; it also contains the SV40 origin of replication, allowing its propagation in mammalian cells expressing the SV40 T antigen. The plasmid expresses the neomycin resistance gene, which allows selection of cells containing the plasmid by growth in the antibiotic G418 (see Note 1). It also contains the mammalian cytomegalovirus (CMV) promoter, with a downstream multicloning site, allowing cloning/expression of a gene of interest in mammalian cells.

The OriP element was obtained from the plasmid pGemoriP (19) as a 2.0-kb RsaI-BamHI DNA fragment. The EBNA-1 gene used in the assays lacks most of the Gly-Ala repeat region (amino acids 101-324), which is not required for the replication and segregation activities of EBNA-1. The fragment containing the EBNA-1 gene was obtained by PCR amplification of the EBNA-1 gene in plasmid p205 (4) using an N-terminal primer containing an Ndel site and a C-terminal primer containing a BamHI site. The amplified fragment was digested with Ndel, the Ndel site was filled in with Klenow, and the fragment was then digested with BamHI.

To generate plasmids used to measure replication and maintenance activities, standard cloning methods were used. Plasmid containing the OriP element

Bglll

Sv40 Repocation Plasmid

Hind 111 Kpn I BamH I EcoR I

EcoRV

SV40 Neomycin resistance Poly A

Fig. 1. pcDNA3, the backbone vector for the plasmids used in the transient replication and long-term maintenance assays. The EBV OriP sequence was cloned into the BgllI and NruI sites of pcDNA3 to generate pc3oriP. The EBNA-1 gene was cloned into the HindlH and BamHI sites located in the multiple cloning site (MCS) of pc3oriP to give pc3oriPE.

Bglll

Hind 111 Kpn I BamH I EcoR I

EcoRV

SV40 Neomycin resistance Poly A

Fig. 1. pcDNA3, the backbone vector for the plasmids used in the transient replication and long-term maintenance assays. The EBV OriP sequence was cloned into the BgllI and NruI sites of pcDNA3 to generate pc3oriP. The EBNA-1 gene was cloned into the HindlH and BamHI sites located in the multiple cloning site (MCS) of pc3oriP to give pc3oriPE.

(pc3oriP) (20) was constructed by ligation of the BamHI-RsaI OriP fragment into the Bglll and Nrul sites of pcDNA3. To construct pc3oriPE (20), which contains both OriP and the EBNA-1 gene, the Ndel (filled-in)-BamHI fragment consisting of the EBNA gene was inserted between the Hindlll (blunted by mung bean nuclease digestion) and BamHI sites of pc3oriP (see Note 2). Ligation products for each construct were transformed into E. coli DH5a, and the resulting cells were plated on LB/ampicillin plates. Ampicillin-resistant colonies were grown in LB/ampicillin medium, and plasmids were isolated by standard miniplasmid purification methods and screened for positive clones through restriction enzyme digestion and DNA sequencing. Plasmids representing the right clones were retransformed into DH5a and purified by a large-scale plasmid purification method that would yield clean transfectable DNA (such as CsCl gradient or Qiagen maxiplasmid purification).

3.1.2. Introduction of Plasmids Into Human Cells

Described below is the introduction of the EBV-based plasmids used in the transient replication and maintenance assays into C33A cells by calcium phosphate transfection (see Note 3).

C33A is a human cervical carcinoma cell line that grows as adherent cells.

1. Grow C33A cells in culture plates in DMEM supplemented with 10% FBS, until cells are 80% confluent.

2. To split cells, remove medium, wash cells with PBS, add trypsin to the cells, and incubate at 37°C until cells lift off the plates.

3. Replate 1/6 of the cells in tissue culture plates containing fresh medium.

4. Repeat when cells are 80% confluent (usually every 2 d).

3.1.2.2. Transfection of C33A

1. At 24 h prior to transfection, plate 1 x 106 cells in a 10-cm tissue culture dish with 10 mL of DMEM/FBS. It is a good idea to plate enough dishes such that the assays can be conducted in duplicate for each plasmid sample to ensure reproducibility of the results.

2. For the transient replication assay, mix 10 |g of pc3oriP or pc3oriPE with 50 ||L 2.5 M CaCl2 and 450 |L dH2O. For the plasmid maintenance assay, do the same using 1 | g of the plasmids.

3. Add the 500-|L mixture from step 2 to 500 |L of 2X HBS, and mix by pipeting up and down seven times.

4. Incubate the resulting mixture at room temperature for 25 min.

5. Add the 1-mL mixture dropwise to the cells from step 1, and incubate for 16 h in a 37°C/5% CO2 incubator. The precipitate in the cell medium should become visible within 10 min of incubation.

6. After the 16-h incubation, remove medium containing the precipitate, wash the cells twice with PBS, trypsinize, and transfer to 15-cm plates containing 20 mL fresh medium. Add 400 mg/mL G418 to the plates that will be used for the plasmid maintenance assay. Place all plates back into the incubator.

3.1.3. Growth of the Transfected Cells

1. Replication and maintenance assays.

a. For the transient replication assay, grow C33A cells transfected with pc3oriP or pc3oriPE for 72 h post transfection.

b. For the plasmid maintenance assay, grow C33A cells transfected with pc3oriP or pc3oriPE for 2 wk post transfection. Change media/G418 selection every 3 d. Cells lacking the plasmids will start to die off within 4-5 d of growth in G418. If G418-resistant cells within colonies start to crowd, then harvest cells by trypsinization, and replate all the cells to spread them out.

2. At the end of the growth period, harvest and count cells (use a hemocytometer).

a. For the transient replication assay, remove as many cells as possible, ensuring that equal cell numbers are obtained from pc3oriP- and pc3oriPE-transfected plates.

b. For the plasmid maintenance assay, remove 5 x 106 cells from pc3oriP- and pc3oriPE-transfected plates (see Note 4).

Pellet cells by centrifugation at 350 g, and wash with PBS.

3.1.4. Extraction and Digestion of Plasmids

Only some of the cells harvested for the transient replication assay will contain the plasmid of interest, since these cells were not placed under G418 selection. The number of cells containing the plasmid will be dependent on trans-fection efficiency and on whether the plasmid can replicate in the cells. Cells harvested for the plasmid maintenance assay will contain the plasmid of interest, since these cells were placed under G418 selection. However, the plasmid can either be maintained as an episome or can be integrated into the C33A genome. The plasmid will integrate in cells where it cannot either replicate or segregate efficiently. A plasmid that can replicate and segregate in cells can be recovered from the cells in both the replication and maintenance assays. To determine whether pc3oriP and pc3oriPE can transiently replicate and be maintained in C33A, these plasmids are extracted from the harvested cells by Hirt's extraction method (21) as described below.

1. Resuspend cells harvested in Subheading 3.1.3. in 350 ||L of PBS.

2. Add 350 ||L of 2X Hirt's solution, and incubate at room temperature for 10 min.

3. Add 140 |L 5 M NaCl to the 700-|L lysate, mix by gently inverting the tube 10 times, and incubate overnight at 4°C.

4. Centrifuge the lysate for 30 min at top speed in a microcentrifuge at 4°C.

5. Keep the supernatant and extract with an equal volume of phenol/chloroform and then with chloroform; in each case, keep the upper layer.

6. Add absolute ethanol (twice the volume of the upper layer), mix, and incubate overnight at -20°C to precipitate DNA.

7. Pellet DNA for 30 min at top speed in a microcentrifuge at 4°C.

8. Resuspend the pellet in 300 |L 0.3 M NaOAc, pH 7.5, and then add 600 |L of absolute ethanol and incubate at -20°C overnight to reprecipitate the DNA.

9. Pellet DNA as in step 7, and dry the pellet at 37°C.

10. To linearize the plasmid DNA with a unique enzyme (for Southern analysis), resuspend in the pellet in: 17 |L 10 mM Tris-HCl, pH 8.5, 2 |L Xhol restriction digest buffer (NEB buffer 2), 1 |L Xhol (20 U; NEB). Incubate at 37°C for 4 h.

11. To the digestion mixture, add 1 |L Dpnl (20 U; NEB) to remove nonreplicated pc3oriP or pc3oriPE. lncubate further at 37°C for 3 h.

12. Add DNA loading buffer, and load onto 0.8% TAE agarose gel. Also include 100 pg of linearized pc3oriP and/or pc3oriPE on the gel as marker. Run the gel overnight at 30 V.

3.1.5. Determining Replication and Maintenance of Plasmids by Southern Blotting

1. Place the gel in 0.25 N HCl depurination buffer, and gently shake on a rocker for 10 min; rinse with dH2O and then incubate in 0.4 N Na0H/0.6 M NaCl denatura-tion buffer for 30 min with shaking.

2. Transfer the DNA samples in the gel to a nylon membrane (GeneScreen Plus, NEN Life Sciences) using standard Southern blotting techniques (see Note 5) (22).

3. Rinse the membrane in 2X SSC, and incubate with Denhardt's blocking buffer for 2 h at 42°C in a hybridization oven.

4. During the 2-h incubation, radiolabel 300 ng of linearized pc3oriP with [a-32P]dCTP using standard techniques for random oligonucleotide primed synthesis (22). Determine the counts per minute (cpm)/mL of the probe.

5. After the 2-h incubation, discard the blocking buffer, and incubate the nylon blot in 10 mL of fresh blocking buffer containing 5 x 106 cpm of the probe that has been denatured (by incubation at 100°C for 5 min followed by incubation on ice for 5 min). Incubate overnight at 42°C in the hybridization oven.

6. Wash the probed nylon: (1) twice with 100mL of 2X SSC/1% SDS for 10 min at room temperature; (2) twice with 100 mL of 2X SSC/1% SDS for 30 min at 65°C; (3) once with 100 mL of 0.2X SSC/0.1% SDS for 10 min at room temperature.

7. Wrap the blot in Saran wrap, and expose on film or a phosphoimager screen for qualitative or quantitative (quantify by ImageQuant software from Molecular Dynamics) measurements, respectively, of the replication and maintenance capabilities of the plasmids. In the experiment described here, the negative control pc3oriP should not be recovered in the transient replication and plasmid maintenance assays, whereas the positive control pc3oriPE should be recovered in both the assays, giving a signal comparable to the 100-pg marker if the transfection was efficient (Fig. 2).

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