Regulation of Transgene Expression in Genetic Immunization

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Brazilian Journal of Medical and Biological Research (1999) 32: 155-162 Regulation of transgene expression 155 ISSN 0100-879X

Regulation of transgene expression in genetic immunization

J.S. Harms1, 1Department of Animal Health and Biomedical Sciences, University of Wisconsin, S.C. Oliveira2 Madison, WI, USA and G.A. Splitter1 2Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brasil

Abstract

Correspondence The use of mammalian gene expression vectors has become increas- Key words J.S. Harms ingly important for genetic immunization and gene therapy as well as · DNA vaccine AHABS basic research. Essential for the success of these vectors in genetic · Gene therapy 1655 Linden Drive · immunization is the proper choice of a promoter linked to the antigen Regulation Madison, WI 53706 · Transcription of interest. Many genetic immunization vectors use promoter elements USA · Transgene Fax: +1-608-262-7420 from pathogenic viruses including SV40 and CMV. Lymphokines E-mail: [email protected] produced by the immune response to proteins expressed by these vectors could inhibit further transcription initiation by viral promot- Research supported by the Robert ers. Our objective was to determine the effect of IFN-g on transgene Draper Technology Innovation Fund expression driven by viral SV40 or CMV promoter/enhancer and the (No. 135-0546) of the University mammalian promoter/enhancer for the major histocompatibility com- of Wisconsin, Madison. plex class I (MHC I) gene. We transfected the luciferase gene driven Presented at the International by these three promoters into 14 cell lines of many tissues and several Symposium “The Third Revolution species. Luciferase assays of transfected cells untreated or treated with g on Vaccines: DNA Vaccines”, IFN- indicated that although the viral promoters could drive lu- Belo Horizonte, MG, Brasil, ciferase production in all cell lines tested to higher or lower levels than November 3-7, 1997. the MHC I promoter, treatment with IFN-g inhibited transgene expression in most of the cell lines and amplification of the MHC I promoter- driven transgene expression in all cell lines. These data indicate that Received October 5, 1998 the SV40 and CMV promoter/enhancers may not be a suitable choice Accepted November 3, 1998 for gene delivery especially for genetic immunization or cancer cy- tokine gene therapy. The MHC I promoter/enhancer, on the other hand, may be an ideal transgene promoter for applications involving the immune system.

Introduction way, proteins produced through mammalian expression vectors can stimulate humoral or During the past few years, the use of cellular immunity. Intracellular expression mammalian gene expression vectors for new of a non-secreted antigen should specifically methods such as genetic immunization has induce cellular immunity through presenta- become increasingly important. Recent suc- tion by the major histocompatibility com- cess with DNA vaccines indicates their enor- plex class I (MHC I) molecules to cytotoxic mous future potential in diverse fields such T cells. During the immune response, many as infectious diseases, allergy and cancer (1- lymphokines are produced by the various 6). Depending on the antigen processing path- cells involved, including IFN-g, that can act

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on the target cell carrying the transgene. public concern over transfecting DNA ele- Modulation of expression of the transfected ments from pathogenic or tumor-causing vi- gene could occur and, depending on the ruses. BL3-6prmtr is the promoter for the promoter used, increase or decrease trans- cattle major histocompatibility complex class gene expression. Therefore, the proper choice I gene. MHC I is expressed on nearly all of promoter/enhancer linked to the gene of tissues, is critical in immune system commu- interest is critical to the success of DNA nication, and can be up-regulated by lym- vaccinations. phokines such as IFN-g (9). We used the mammalian MHC I pro- The SV40, CMV, and MHC I promoter/ moter/enhancer, BL3-6prmtr (7), and com- enhancers have complex cis elements that pared transgene expression and effects of bind varied cellular trans-factors (10-12). IFN-g regulation to the popular viral pro- SV40 and MHC I promoters both have AP2 moter/enhancers of SV40 and CMV in vari- binding sites while CMV and MHC I pro- ous cell lines of several species. To date, moters both have ATF binding sites. In com- almost all commercial mammalian expres- mon to all three promoters are AP1 and sion vectors use viral promoter/enhancer se- NFkB binding sites. Additionally, all three quences from pathogenic viruses such as promoters have interferon response se- simian virus 40 (SV40), or human cytome- quences. However, for CMV and SV40, fac- galovirus (CMV). Although these promoter tors binding to this region can inhibit tran- elements are from pathogenic viruses, they scription (13,14), whereas, for MHC I, inter- have become very useful due to high tran- feron response factors enhance transcription scription initiation ability in most mamma- (15,16). lian tissues (8). However, in genetic immunization applications involving human pa- Comparison of luciferase expression tients or animal husbandry, a mammalian driven by MHC I, SV40, and CMV promoter may be more desirable for easing promoter/enhancers

Table 1 - Cell lines used for transfections. The luciferase gene driven by BL3-6prmtr (cattle MHC I), SV40, or CMV promoter/ All cell lines were provided by the American Type Culture Collection (ATCC). enhancer was used as a reporter gene transiently transfected in cell lines of various Species Cell line Tissue tissues from several species (listed in Table

Mouse A20.J B lymphoma 1). Assays of luciferase production were C2C12 Myoblast used to compare the relative strengths of the G8 Myoblast MHC I promoter/enhancer to the viral pro- P815 Mastocytoma moter/enhancers transgene expression abili- RAW 264.7 Macrophage ties in each cell line. Each of the three pro- Human COLO 320HSR Colon carcinoma moters was able to drive luciferase expres- LCL 721.221 B lymphoblast SW 837 Rectal carcinoma sion in all cell lines tested. However, BL3- 6prmtr was, in many cases, weaker than Cow BL3.1 B sarcoma MDBK Kidney either SV40 or CMV promoters. The data, summarized in Table 2, show that SV40 was Dog D17 Osteocarcinoma stronger than BL3-6prmtr in 12/14 cell lines Monkey Vero Kidney and CMV was stronger in 6/14 cell lines. Rat NMU Mammary carcinoma Comparisons between SV40 and CMV pro-

Sheep FLK Kidney moter/enhancers could not be made because the vector constructs were not similar.

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Effect of IFN-g addition to transient Table 2 - Effect of IFN-g on luciferase transgene production driven by MHC I, SV40, or transfected cell culture on transgene CMV promoters. promoter Note: Increased expression after IFN-g treatment is in bold numbering. Decreased expression after IFN-g treatment is in italics numbering. aThree transfections per cell Luciferase production was positively or line of each luciferase gene construct (in parentheses) driven by MHC I, SV40, or CMV negatively altered by IFN-g treatment de- promoters were done for each experiment. Transfections were performed using the cationic lipid method (LipofectAMINE; Life Technologies, Inc., Gaithersbourg, MD). At pending on the promoter and cell line. Tran- least three experiments were done for each cell line. Transfection efficiency was siently transfected cell cultures were treated measured using internal controls for either ß-galactosidase (pGL6 and pGL2 Control) or g CAT (p6/Luc and pcDNA3/Luc). bIFN-g was added (+) or not added (-) to transient or not treated with rIFN- for 16 h and then transfects which were assayed for luciferase production 16 h later. Human rIFN-g was assayed for luciferase production. Results added to human, cow, dog, monkey, and sheep cell lines while mouse rIFN-g was demonstrate that BL3-6prmtr-driven lu- added to mouse and rat cell lines (see Table 1). Numbers represent mean picogram (pg) luciferase per transfection calculated using a luciferase standard curve. The ciferase production increased in all cell lines, coefficient of variation was less than 10% for each experiment. whereas the SV40 or CMV promoter-driven luciferase production decreased in most cell Cell line MHC I SV40 CMV lines (Table 2). (pGL6)a (p6/Luc) (pGL2 Control) (pcDNA3/Luc) Using the mammalian expression vectors b p6/Luc (MHC I promoter driven) and (-) (+) (-) (+) (-) (+) (-) (+) pcDNA3/Luc (CMV promoter driven), we A20.J 30 41 50 81 826 409 126 16 established stable transfects of several cell BL3.1 40 54 7 10 8 5 4 2 52 61 g C2C12 19 20 403 83 182 87 lines to ascertain whether IFN- would af- COLO 56 107 30 53 28 11 11 2 fect chromosome integrated promoter/trans- D17 38 40 14 16 88 68 30 22 gene expression differently than episomal FLK 36 251 99 234 215 354 59 89 G8 28 96 9 17 810 209 53 23 transgene expression. We isolated neomy- 721.221 34 39 19 20 43 18 18 17 cin-resistant clones of A20.J, P815, RAW MDBK 8 9 8 10 10 9 13 8 NMU 24 63 91 176 250 75 154 34 264.7, COLO 320HSR, SW 837, and BL3.1 P815 28 30 4 5 57 34 4 2 cell lines and selected the highest luciferase- RAW 20 74 12 25 50 176 9 11 expressing clones of each transgene con- SW837 8 9 9 10 16 12 8 7 Vero 34 62 14 35 208 150 10 7 struct. IFN-g treatment of these stable transfects produced similar results (Table 2) us- g ing the same IFN- dosage (500 U/ml) and C2C12 G8 measurement time point (16 h; data not 180 450 shown). 160 400 Many gene delivery vaccination applica- 140 350 120 300 tions use muscle as the target tissue (17-19). 100 250 Our results indicate that while SV40 and 80 200 CMV promoters drive luciferase expression 60 150 40 100 in myoblasts to higher levels than BL3- 20 50

g Fentograms luciferase 6prmtr, addition of IFN- drastically reduces 0 0 SV40- and CMV promoter-driven expres- -IFN +IFN -IFN +IFN pGL2 Basic pGL6 p6/Luc sion while increasing BL3-6prmtr-driven pGL2 Control pcDNA3/Luc expression. Figure 1 gives a graphic illustra- tion of this change. Figure 1 - BL3-6prmtr, SV40, and CMV promoter-driven luciferase production in transfected myoblasts. The mouse myoblast cell lines, C2C12 and G8, were transfected with luciferase g gene constructs driven by no promoter (pGL2 Basic), the SV40 promoter/enhancer (pGL2 Kinetics of IFN- -treated stable transfect Control), the CMV promoter/enhancer (pcDNA3/Luc), or the cattle MHC class I promoter/ luciferase production enhancer (pGL6; p6/Luc). Transfectant cell cultures were either untreated (-IFN) or treated (+IFN) with IFN-g for 16 h and then assayed for luciferase production. Three experiments of three transfections of each luciferase construct were performed. Measurements represent IFN-g dosage and kinetics of luciferase the mean relative light units (RLU) with coefficient of variation of less than 10%.

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expression driven by CMV or MHC I pro- synergistic, or antagonistic effects on the moters in these stable transfects were meas- transgene promoter. For example, tumor ne- ured. These data showed an optimal IFN-g crosis factor-alpha (TNF-a), another lym- dosage of 100-500 U/ml for both CMV and phokine produced during an immune re- MHC I promoters for highest luciferase pro- sponse, has been shown to inhibit CMV duction at all time points (data not shown). transcription but acts synergistically with The kinetics of luciferase transgene produc- IFN-g in enhancing MHC I transcription tion showed that the amount of CMV-driven (20,21). Using the supernatant from mito- luciferase product peaked at approximately gen-stimulated T cells as a source of immune 8 h and then dropped to below baseline response lymphokines, we tested transgene (untreated) levels at 16-24 h, while the MHC expression in response to these mixed lym- I-driven luciferase product continued to rise phokines as well as IFN-g in a transiently through 48 h. Figure 2 shows these results transfected human colorectal carcinoma cell using stably transfected human colon carci- line. The results, shown in Figure 3, indicate noma (COLO 320HSR) and mouse macro- that the T cell blast supernatant increased phage (RAW 264.7) cell line clones. transgene expression driven by MHC I promoter to an even greater extent than IFN-g Effect of mixed lymphokine addition to alone. Viral promoter-driven expression was transfected cell culture on transgene up-regulated by the mixed lymphokines com- expression pared to IFN-g-treated cells but not to the level of untreated cells. An in vivo immune response that was either initiated or promoted by the transgene Discussion would release a multitude of lymphokines, along with IFN-g, that may have additive, Foremost in DNA vaccine delivery vector design should be the proper choice of Figure 2 - Kinetics of luciferase transgene expression in stably 120 promoter/enhancer to drive expression of transfected cell lines. Human co- the transferred gene. This choice is depend- 100 lon carcinoma (COLO 320HSR) ent upon target cell type as well as the func- or mouse macrophage (RAW 80 264.7) cell clones with a chro- tional goal of the transgene. DNA vaccine mosome-integrated luciferase 60 applications are designed to affect immune transgene driven by either the function to protect against a pathogenic agent, CMV promoter (CMV/Luc) or the 40 COLO [CMV/Luc] or lymphokine gene transfer to fight cancer. MHC I promoter (MHC I/Luc) 20 were cultured in 24-well plates Fentograms luciferase COLO [MHCI/Luc] These applications stimulate the immune and treated with IFN-g (500 U/ 0 system resulting in production of many lym- ml) for 0, 4, 8, 24, 48, and 72 h. 048244872 Cells were harvested in 100 µl Time (h) phokines that can directly act on the trans- lysis buffer at these time points 180 fected target cell and thus indirectly act on o and lysate was stored at -70 C 160 the transgene promoter. At least one of these until assayed for luciferase. 140 lymphokines, IFN-g, is a potent antiviral Graphs show the mean relative 120 light units (RLU) and standard de- agent, and has been shown to inhibit viral 100 viations of four experiments. transcription of SV40 and CMV (13,14), 80 60 RAW [CMV/Luc] although many promoters for transgene ex- 40 RAW [MHCI/Luc] pression are from SV40 or CMV (2,17-19,22-

Fentograms luciferase 20 25). Our objective was to determine what g 0 effect IFN- had on transgene expression 048244872 Time (h) driven by SV40, CMV, or MHC I promoter/ enhancers using cell lines of various tissues

Braz J Med Biol Res 32(2) 1999 Regulation of transgene expression 159 to ascertain the best choice for gene delivery ciferase from transfect cultures of CMV pro- vector promoter in immunoregulatory appli- moter-driven transgene were near or below cations. baseline levels at this time. These experi- Our results show a down-regulation ef- ments showed an initial increase in CMV fect of IFN-g on both SV40 and CMV pro- promoter-driven production peaking at moter/enhancer-driven transgene in most cell around 8 h and then dropping to below lines tested and an up-regulation effect of baseline levels. Also, after 48 h, MHC I IFN-g on the MHC I promoter/enhancer- promoter-driven transgene product levels driven transgene in all cell lines tested. We began to drop. We believe that these results note, however, that even with IFN-g down- were due to the cells being plated at 70% regulation, viral promoter-driven expression confluence at the beginning of the experi- was frequently greater than that of IFN-g up- ment, and to the fact that the medium was not regulated MHC I promoter-driven expres- changed through the 72-h period. Therefore, sion. Nonetheless, these negative selective the slight increase in CMV promoter-driven pressures may shorten the expressible life of luciferase production may reflect increased the transgene. For example, we noticed in cell numbers to confluence rather than an our stably transfected cell clones that lu- IFN-g-induced enhancement. The IFN-g in- ciferase expression driven by BL3-6prmtr hibitory effect on the CMV promoter and stayed at the same level for many cell pas- enhancing effect on the MHC I promoter sages whereas luciferase expression driven probably occurred within 4 h. The reason by CMV promoter showed a dampening MHC I promoter-driven luciferase produc- of expression soon after cloning. This theory tion began dropping by 48-72 h may have has yet to be tested. been due to lack of fresh medium (growth In fact, studies using the stable transfects factors) in the culture and perhaps the begin- showed an ever increasing amount of lu- ning of cell death. Since luciferase is a fairly g ciferase from MHC I promoter-driven trans- stable protein, these data indicate that as Figure 3 - Effect of IFN- and T g cell blast supernatant (sup) on gene over 48-72 h while amounts of lu- long as IFN- is present, the MHC I promoter BL3-6prmtr, SV40, and CMV promoter-driven luciferase pro- COLO 320HSR duction in transfected human 9000 colorectal carcinoma cells. The human colorectal carcinoma cell pGL2 Basic line COLO 320HSR was trans- 8000 fected with luciferase gene con- pGL2 Control structs driven by no promoter 7000 (pGL2 Basic), the SV40 promoter/enhancer (pGL2 Control), pGL6 6000 the HCMV promoter/enhancer (pcDNA3/Luc), or the cattle MHC class I promoter/enhancer pcDNA3/Luc 5000 (pGL6; p6/Luc). Transfected cell cultures were untreated or 4000 p6/Luc treated with either IFN-g (+IFN) or 10% supernatant from 3d mi- togen-stimulated T cells (+T

Fentograms luciferase 3000 Blast sup) and then assayed for luciferase production. Three ex- 2000 periments of three transfections of each luciferase construct 1000 were performed. Measure- ments represent the mean relative light units (RLU) with a coef- 0 Untreated+IFN +T Blast sup ficient of variation of less than 10%.

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is up-regulated and the CMV promoter is did not alter the general effect of IFN-g on a down-regulated. particular cell line according to our stable or Although the type of cell or tissue is transient transfection results using pcDNA3/ important in determining whether IFN-g as Luc and p6/Luc. well as other immune response lymphokines We still need to elucidate whether these will enhance or inhibit transgene expression data represent effects of an actual immune driven by the viral promoters, our results response to the transgene typical in DNA indicate that most probably expression will vaccination. Intramuscular DNA vaccina- be down-regulated. The MHC I gene, on the tion of mice with rabies virus glycoprotein other hand, is a critical immune system com- gene driven by SV40 or MHC I promoter munication molecule and has been shown to developed a comparable antibody response have enhanced transcription due to lympho- (28). However, co-inoculation of mouse IFN- kines produced during an immune response g plasmid and either of the SV40- or MHC I- (9). The use of T cell blast supernatant- driven rabies vaccine vectors resulted in a treated transfected cell lines confirmed the reduced antibody and T helper cell prolifera- enhanced expression of BL3-6prmtr-driven tion response in these mice. Cattle vacci- transgenes, similar to the results shown in nated with bovine herpesvirus-1 glycopro- Figure 3, for all cell lines tested, including tein D (gD) gene driven by CMV or MHC I COLO 320HSR, LCL 721.221, BL3.1, and promoter delivered by the biolistic process FLK (data not shown). resulted in a greater gD-specific antibody The MHC I promoter/enhancer was used response to the CMV/gD vaccine compared to drive luciferase in two different promoters to the MHC I/gD vaccine (data not shown). and, as seen in Table 2, caused different In both of these examples the measurement luciferase expressions. Vectors pGL2 Con- of the DNA vaccine response was based on trol (SV40 driven) and pGL6 (MHC I driven) antibody titer or T helper cell proliferation - were standard reporter gene vectors typical immune responses that depend upon MHC II for use in transient transfection assays. Vec- antigen presentation. Therefore, the protein tors pcDNA3/Luc (CMV driven) and p6/ produced by the transgene had to be secreted Luc (MHC I driven) were engineered from a by the transfected cell, taken up and pro- mammalian expression vector containing a cessed by antigen presenting cells, and pre- separate, selectable neomycin gene independ- sented in the context of MHC II to B and T ently driven by the SV40 promoter/enhancer. helper cells. To truly assess the efficacy of This vector is typical for use in stable trans- the MHC I versus the viral DNA vaccine fections. Even though the same MHC I pro- promoters in vivo, studies must be done on moter/enhancer drove expression of the same the long-term or memory response. Our data luciferase gene, the lower expression results suggest that, although MHC I is a weaker of p6/Luc compared to pGL6 could be due to promoter than the viral promoters, immune transcription factor competition by the SV40 response cytokines could eventually shut promoter or some inhibitory effect by the down the viral promoter while augmenting neomycin gene product, as others have shown the MHC I promoter. Thus there may be long (26,27). The fact that many selectable marker term protection with DNA vaccines driven genes in mammalian gene expression vec- by the MHC I promoter. Additionally, stud- tors are under viral promoter control should ies must be done on cytotoxic T cell re- be another consideration in choosing the sponses of MHC I versus viral promoter- best gene delivery vector for immune re- driven vaccines. Because the cytotoxic T sponse applications. Whether the transgene cell response involves antigen presentation was integrated in the genome or episomal by MHC I (most likely in the transfected

Braz J Med Biol Res 32(2) 1999 Regulation of transgene expression 161 cell), the lower but longer term expression of lived expression of these transgenes. Our MHC I promoter may induce a superior cy- results offer an explanation for these obser- totoxic T cell response compared to viral vations. Admittedly, the viral promoters of promoter-driven vaccines. SV40 and CMV are generally stronger than We believe the MHC I promoter/en- the MHC I promoter, BL3-6prmtr. Never- hancer, BL3-6prmtr, would be a better choice theless, our data suggest that for long-term for gene delivery vector promoter in immu- sustained transgene expression necessary for noregulatory applications than the widely an optimal immune response, the mamma- used viral promoters of SV40 and CMV. lian MHC I promoter/enhancer could be a This belief is based on the generally inhibi- preferable option. tory effect of IFN-g, as well as other lymphokines, on SV40 and CMV promoter- Acknowledgments driven transgene expression, and the ampli- fied effect of IFN-g and other lymphokines We thank the Instituto de Ciências on MHC I-driven transgene expression. Many Biológicas, UFMG, especially in the person researchers using viral promoters to express of Dr. Vasco Azevedo, for his effort in mak- transgenes have been frustrated by the short- ing this conference possible.

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