Data Analysis

The addition of the RL (Cap-mediated) signal in the bicistronic system allows for the simultaneous normalization of transfection efficiency and M-AS specificity within each transfection set. The relative translation efficiency of IRES-mediated FL values is determined by comparing the averaged FL/RL ratios of M-AS-treated to nontreated controls for each transfection set (e.g., transfection 1 vs transfection 2). Values obtained are plotted as percent inhibition for each transfection set (Fig. 1). Comparison of inhibition profiles showed that M-AS reduced FL levels in all transfection sets, but to varying levels.

MTT data were analyzed individually for each transfection. Values for each transfection were averaged. Comparison of cells-only controls to the mock-

Fig. 2. Inhibition of cell viability. MTT values were averaged for each transfection, and cellular toxicity was determined by direct comparison of transfections with the mock-transfection controls.

transfected controls (transfections 13 and 14) showed no difference in MTT values, suggesting that the addition of transfection components to cells was well tolerated (data not shown). Subsequently, these mock-transfected values (arbitrarily set to 100%) were directly compared with actual transfection values. Values are plotted as a percentage of the mock-transfected control values (Fig. 2). Comparison of results within each transfection set showed no differences in MTT values, demonstrating that the 3 ^M M-AS test level was not toxic to cells. However, comparison of transfection sets with each other showed varying levels of inhibition. Sets 1-3 showed little or no inhibition, whereas sets 4-6 exhibited a more dramatic reduction in MTT levels compared to controls. Comparison of the time constant values showed an inverse relationship between time constant levels and cytotoxicity (Table 1). Moreover, this increase in time constant values was directly associated with increased M-AS efficacy (Table 1 and Fig. 1). The transfection parameters used for set 3 produced the greatest inhibition levels (>95%) with only minor (<10%) cyto-toxicity.

Use of the optimal transfection procedures can subsequently be used in more elaborate assays including controls. Therefore, an expanded assay was performed using increasing concentrations of HCVm330-354 or the randomized control, RDMm330-354, and 0.25-kV/960-^F electroporator settings. Values were plotted in a fashion identical to Fig. 1. The results demonstrated that HCVm330-354 produced a dose-dependent inhibition of FL (IRES) values (Fig. 3). Inhibition of the samples containing RDMm330-354 showed minor but not dose-dependent inhibition, further supporting the specificity of HCVm330-354 toward the HCV IRES.

Fig. 3. Dose-dependent inhibition of the HCV IRES. The authentic antisense HCVm330-354 (open bars) or randomized control RDMm330-354 (shaded bars) were added to separate transfections at increasing concentrations. Transfection conditions were identical for all assay conditions (0.25kV/960 ^F). Percent inhibition profiles were calculated in a fashion identical to those in Fig. 1.

Fig. 3. Dose-dependent inhibition of the HCV IRES. The authentic antisense HCVm330-354 (open bars) or randomized control RDMm330-354 (shaded bars) were added to separate transfections at increasing concentrations. Transfection conditions were identical for all assay conditions (0.25kV/960 ^F). Percent inhibition profiles were calculated in a fashion identical to those in Fig. 1.

These data demonstrated that electroporation can be a valuable approach for introducing nonionic M-AS into HeLa cells in culture. The use of a bicistronic plasmid simplified the assay format while providing a valuable control for transfection efficiency and specificity. Furthermore, the concomitant analysis of cytotoxicity by MTT assay demonstrated that limiting cytotoxicity should greatly enhance the inhibition profile of a given M-AS, which is a critical aspect of selecting candidate AS-ODNs for further development.

4. Notes

1. Whenever reporter assays are used, it is essential to check for nonspecific activity against the specific reporter gene before performing elaborate studies. The use of reporter controls (e.g., pCMV-FLUC and pCMV-RLUC) should be analyzed prior to elaborate transfection assays to ensure that M-AS does not produce undesirable nonspecific effects. Furthermore, the use of random or mismatch controls will lend greater confidence of M-AS specificity.

2. This described protocol uses M-AS. However, it should also be useful for trans-fection studies using other nonionic antisense chemistries including peptide nucleic acids.

3. Cell-based assays require plasmids that possess a promoter element that is active within the cell line of choice. Plasmid DNA should be prepared by a method such as column purification (Maxi-kit; Qiagen, Valencia, CA) and eluted into endot-oxin-free sterile H2O. Poorly prepared DNA can have extremely adverse effects on transfection.

4. The HeLa cells used in this study were Texas Hela cells received from Dr. C. Guantt, University of Texas Health Science Center, San Antonio. Substitution of another adherent Hela line should produce comparable results.

5. The cell pellet should first be resuspended in a minimal volume of <5.0 mL. Trying to resuspend the cell pellet in a large volume will result in cell clumps remaining in solution.

6. Work quickly. Cells that remain on ice for extended periods of time can settle out into the bottom of the cuvet, which can adversely affect transfection efficiency.

7. Excess moisture on electrodes can result in arcing, potentially damaging the electroporator.

8. Be sure to select a pipet that is long enough to reach the bottom of the cuvet and has an adequate capacity (e.g., 1.0 mL serological pipet [7521; Falcon]).

9. Additional remaining cell volumes can be used to setup additional plates for time course experiments if desired.

10. MTT is very toxic. Handle carefully only with gloved hands and dispose of used solutions properly.

11. To hasten dilution of SDS into the NN-DMF/H2O solution, heat the solution to 37°C.

12. The firefly luciferase/luciferin substrate reaction is strongly influenced by temperature. Preequilibration to room temperature ensures that the substrate will not change temperature during the course of the assay.

13. We used the dual injection system to prevent adverse effects of reporter decay when assaying an entire plate. As an alternative, samples can be assayed individually in a single-format luminometer. In addition, newer substrate mixes that have increased stability are commercially available, which may alleviate the need for injectors (Promega).

14. The dual luciferase assay must be performed sequentially. FL must be assayed first, followed by RL. Be sure that injectors are primed properly before starting the assay.

References

1 Choo, Q. L., Kuo, G., Weiner, A. J., Overby, L. R., Bradley, D. W., and Houghton, M. (1989) Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science. 244, 359-62.

2 Brown, E. A., Zhang, H., Ping, L. H., and Lemon, S. M. (1992) Secondary structure of the 5' nontranslated regions of hepatitis C virus and pestivirus genomic RNAs. Nucleic Acids Res. 20, 5041-5045.

3 Jubin, R. (2001) Hepatitis C IRES: translating translation into a therapeutic target. Curr. Opin. Mol. Ther. 3, 278-287.

4 Hanecak, R., Brown-Driver, V., Fox, M. C., et al. (1996) Antisense oligonucleotide inhibition of hepatitis C virus gene expression in transformed hepatocytes. J. Virol. 70, 5203-5212.

5 Jubin, R., Vantuno, N. E., Kieft, J. S., et al. (2000) Hepatitis C internal ribosome entry site (IRES) contains a phylogenetically conserved GGG triplet essential for translation and IRES folding. J. Virol. 74, 10,430-10,437.

6 Summerton, J., Stein, D., Huang, S. B., Matthews, P., Weller, D., and Partridge, M. (1997). Morpholino and phosphorothioate antisense oligomers compared in cell-free and in-cell systems. Antisense Nucleic Acid Drug Dev. 7, 63-70.

7 Kolupaeva, V. G., Pestova, T. V., and Hellen, C. U. (2000) An enzymatic footprinting analysis of the interaction of 40S ribosomal subunits with the internal ribosomal entry site of hepatitis C virus. J. Virol. 74, 6242-6250.

8. Kieft, J. S., Zhou, K., Jubin, R., and Doudna, J. A.. (2001) Mechanism of ribosome recruitment by hepatitis C IRES RNA. RNA 2, 194-206.

9. Sherf, B. A., Navarro, S. L., Hannah, R. R., Wood, K. V. (1996) Dual-luciferase reporter assay: an advanced co-reporter technology integrating firefly and Renilla luciferase assays. Promega Notes 57, 2-9.

10. Gerlier, D. and Thomasset, N. (1986) Use of MTT colorimetric assay to measure cell activation. J. Immunol. Methods 94, 57-63.

0 0

Post a comment