THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1994 by The American Society for ’ Biochemistry and Molecular Biolom, Inc.

Val. 269, No. 47, Issue of November 25, pp. 29903-29907, 1994 Printed in U.S.A.

Dexamethasone Enhancement of Gene Expression after Direct Hepatic DNA Injection*

(Received for publication, March 28, 1994, and in revised form, July 22, 1994)

Robert W. Malone*$, M. Anne HickmanSnll, Karin Lehmann-Bruinsma**, Tracey R. Sih**, Rosemary WalzemSS, Don M. Carlsonll, and Jerry S. Powell**§0 From the Departments of $Medicine:Pathology, IMolecular and Cellular Biology, **Medicine:Hematology and Oncology, and $$Veterinary Medicine:Molecular Biosciences, University of California, Davis, California 95616

The critical physiological functions of the liver make hepatocytes important targets for therapeutic gene de- livery. This study reports significant gene expression following direct injection of plasmid DNA into the livers of rats and cats. Transfection was characterized using luciferase and Lac Z expression from plasmids with the cytomegalovirus immediate early promoter/enhancer (CMV IE) or the Rous sarcoma virus long terminal re- peat (RSV LTR). Dexamethasone treatment enhanced and prolonged transfected gene expression, possibly by activating gene expression. Southern analysis of total DNA extracted from liver at various times following in- jection detected persistent unintegrated plasmid DNA which maintained a prokaryotic methylation pattern. This study demonstrates the feasibility of direct DNA injection in the experimental analysis of hepatic gene expression in vivo.

Multiple approaches have been used for in vivo or in vitro hepatic gene transfer, the majority of which rely on recombi- nant viral vectors (1-31, liposomal delivery vehicles (4-61, or peptide-DNAcomplexes (7,8). These methods generally require partial hepatectomy to stimulate hepatocyte regeneration or to obtain hepatocytes for in vitro manipulation. The need for par- tial hepatectomy substantially increases the morbidity and complexity of these procedures. Gene delivery based on recom- binant viral vectors is further complicated by potential viral- associated toxicities including helper virus production and in- sertional mutagenesis (9, 10). Reporter gene expression after direct injection of DNA in striated muscle has been reported (11, 12), and infectivity was seen after intramuscular and in- trahepatic injection of DNA from cloned hepatitis B and poly- oma virus (13-15). However, several recent reports have sug- gested that liver is not capable of efficient transfection by direct injection of DNA (16, 18).

In this study, we demonstrate successful gene transfer into hepatocytes following direct injection of plasmid DNA into he- patic tissue, and the use of this method for analysis of gene

Medical Center Medical Scholars Grant (to R. W. M.), a Tobacco-related * This work was supported in part by a University of California, Davis

Disease Research Program Award (to J. S. P.), and the American Heart Association, California Affiliate (to J. S. P.). The costs of publication of this article were defrayed in part by the payment of page charges. “his

with 18 U.S.C. Section 1734 solely to indicate this fact. article must therefore be hereby marked “aduertisement” in accordance

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankmIEMBL Data Bank with accession number(s) M21295.

$ Both authors contributed equally to this work. 8 Recipient of a Bank of AmericdGiannini Foundation Fellowship. 11 Recipient of a National Institutes of Health Postdoctoral Fellowship.

To whom correspondence should be addressed. “el.: 916-752-8375; Fax: 916-752-3085.

$5 Burroughs Wellcome Experimental Therapeutics Scholar.

expression in intact animals. Transfected gene expression was characterized by quantitative luciferase protein analysis and qualitative in situ P-galactosidase staining. The experiments were performed in both rat and cat models, demonstrating that gene expression after direct intrahepatic injection is not species specific. Acute hepatic inflammation likely occurred at the site of direct injection and possibly reduced expression from trans- fected genes. To overcome this possible inflammation, animals were treated with dexamethasone, resulting in enhanced and prolonged gene expression. Southern analysis of liver DNA de- tected extended persistence of non-replicating plasmid DNA within the liver tissue, but did not detect chromosomally inte- grated DNA. These findings demonstrate that direct DNA in- jection can result in substantial hepatic gene expression that can be significantly enhanced by dexamethasone treatment.

EXPERIMENTAL PROCEDURES Animals-Male Sprague-Dawley rats (5 weeks to 12 months) were

obtained from Charles River Laboratories. Male specific pathogen-free cats were maintained at the Feline Nutrition and Pet Care Center, University of California, Davis, and ranged in age from 5 to 30 months.

Hepatic Injections-Rats were anesthetized with halothane (5% for induction, 2% for maintenance), and cats with atropine (0.02 mgkg), acepromazine (0.1 mgkg), sodium thiamylal(10 mgkg), and halothane (1.5%). Livers were visualized through ventral midline incisions and 500 pg of plasmid DNA (diluted in 2-3 ml of Dulbecco’s modified Eagle medium, Life Technologies, Inc.) was injected directly into the hepatic parenchyma via a 25-gauge %-inch needle. The needle was inserted into five different sites separated by about 5 mm in a single liver lobe. Medium was injected continuously with enough force to blanche the surrounding parenchyma without causing tissue distention while the needle was inserted and removed. Any bleeding associated with injec- tions was controlled by local pressure, and the abdomen was then closed. To obtain liver tissue for analysis, the animals were either killed or had surgical biopsies performed at 48 h, unless otherwise indicated. Dexamethasone treatment of rats consisted of daily subcutaneous in- jections of 1 mgkg, supplemented with twice daily injections of 125 mgkg chloramphenicol. Cats were treated with daily subcutaneous injections of 0.3 mgkg dexamethasone, with a 1 mgkg pre-treatment dose the day prior to plasmid injection. Cats were also treated twice daily with 20 mgkg ampicillin. Animal care throughout the study was in compliance with the “Guide for the Use and Care of Laboratory Animals” developed by the Institute of Laboratory Animal Resources of the National Research Council. The experimental protocols were ap- proved by the University of California a t Davis, Animal Use and Care Administrative Advisory Committee, utilizing the guidelines provided by the American Association for Laboratory Animal Care.

Plasmids-pCMVL was constructed by subcloning the HindIIIISmaI Photinus pyralis luciferase cDNA fragment from pRSVL (19) into the polylinker of pRc/CMV (Invitrogen, Sorrento Valley, CA). In this plas- mid, luciferase transcription is under the control of immediate early promoter/enhancer sequences from the human cytomegalovirus (CMV IE).’ pRSVL contains the Rous sarcoma virus long terminal repeat

’ The abbreviations used are: CMV, cytomegalovirus; IE, immediate early promoter/enhancer sequence; X-gal, 5-bromo-4-chloro-3-indolyl P-D-galactopyranoside; RSV, Rous sacroma virus; LTR, long terminal repeat; GRE, glucocorticoid response element.

29903

29904 Demmethasone Enhancement of Dansfected Gene Expression controlling the expression of the luciferase cDNA. pCMVp consists of CMV IE linked to a modified Escherichia coli lac 2 open reading frame and was purchased from Clontech (Palo Alto, CA).

p-GuZuctosiduse Staining-Livers were perfused in situ for 30 min- utes with 2% paraformaldehyde, 0.1 M pipes, 2 m~ MgCZ, and 1.25 m~ EGTA. The livers were removed, blocked, and equilibrated in phos- phate-bdered saline, 2 IUM MgCl,, and 30% sucrose at 4 "C. ARer freezing in OCT embedding compound (Miles Incorporated Diagnostic Division, Elkhart, IN), 5 - p cryostat sections were sampled, placed on polylysine-coated slides, and stained with 5-bromo-4-chloro-3-indolyl @+galactopyranoside (X-gal) as described elsewhere (20). After X-gal staining, tissue sections were lightly counterstained with hematoxylin and eosin.

Luciferase Quantitution-Liver extracts were prepared by Dounce homogenization after addition of 4 volumes of cold buffer (0.1 M potas- sium phosphate, pH 7.8, 1 m~ dithiothreitol, and 1% Triton X-100). Tissue homogenates were c l d e d at 4 "C in an Eppendorf microcen- trifuge at maximum speed for 5 min, and the supernatants kept frozen at -20 "C until analysis. Luciferase activity was measured using an Analytical Luminescence 2010 Luminometer with photon detection in- tegrated over 15-s intervals (11). Conversion from light units to lucif- erase protein mass was calculated from standard curves based on purified luciferase protein standards (Analytical Luminescence Labe ratories). Samples of the protein standard were diluted into cytosols of normal liver. Two fg of luciferase protein could be detected, and a linear dose response was observed (2 fg to 100 pg; relative light units = 9255 (pg of luciferase) + 60.8, ? = 0.99).

Cell Culture-Primary hepatocytes were isolated from rats following in situ perfusion of the liver (21) and were plated on type IV collagen coated plates (Collaborative Research, Inc.). Cultured hepatocytes were transfected 4-5 h after isolation with pCMVL, pRSVL, or pCMV/3 using cationic liposomes. The liposomes were prepared by evaporating a solution of dioctadecylamidoglycy1spermine:dioleoylphosphatidyl- ethanolamine (50:50) in chloroform to form a thin film, hydrating the film overnight in deionized water, and then sonicating the resulting emulsion to clarity. Final lipid concentration was 1 mg/ml. Dioctadecyl- amidoglycylspermine was obtained from Pmmega (Madison, WI) and dioleoylphosphatidylethanolamine was obtained from Sigma. Plasmid DNA (1 pg) and 50:50 dioctadecylamidog1ycyLspermine:dioleoylphos- phatidylethanolamine liposomes (7.5 pg) were diluted separately in 125 pl of Dulbecco's modified Eagle's medium and then mixed. The DNA lipid mixture (250 pl) was added to each well of a 24-well microtiter plate (1 x lo5 hepatocytedwell). ARer 45 min, 750 pl of minimum essential medium (Life Technologies, Inc.) containing 10 pg/d insulin, 10 Mml glucagon, 50 ng/ml epidermal growth factor, 100 milliunitdml human growth hormone, 100 ng/ml prolactin, 2 x lo4 M selenium, 100 IU/ml penicillin, and 100 pg/ml streptomycin, were added to each well with or without 500 n g / d dexamethasone. Medium was replaced daily following transfection. At 48 h, cells were lysed, and total luciferase activity was measured. Six to 10 replicates of each treatment group were performed. In separate experiments, hepatocytes were prepared from five animals and analyzed in a similar fashion.

Southern Analysis-DNA was isolated from feline liver homogenates prepared for luciferase analysis. Both chromosomal and low molecular weight (extra-chromosomal) DNA were isolated by the method of Hirt (22) and analyzed for size and methylation pattern. The isoschizomer restriction enzymes NdeII and DpnI were used to cleave the extra- chromosomal DNA preparation prior to Southern blot analysis. NdeII will not cleave DNA which is methylated, whereas DpnI will cleave only if the sequence retains a bacterial methylation pattern. Bacterial plasmid DNA acquires a eukaryotic methylation pattern upon replica- tion in eukaryotic cells. Twenty pg of either chromosomal or extra- chromosomal DNA were digested with restriction enzymes, and DNA fragments were separated by electrophoresis in 0.7% agarose in 1 x Tris-acetate-EDTA. Gels were blotted in 0.4% NaOH and 1.5% NaCl onto Hybond-N nylon membranes for 18 h. Filters were washed briefly in 2 x SSC, baked at 80 "C for 2 h, and prehybridized in 0.2 M sodium phosphate (pH 7.2), 7% SDS, and 1 IUM EDTA(23). DNAfragments were hybridized with a probe prepared by labeling a 1300-base pair EcoFtI DNA fragment of pCMVL with [C+"]dCTF' by random priming. After hybridizing overnight a t 65 "C, filters were washed three times for 15 min each time, in 40 IUM sodium phosphate (pH 7.2),1% SDS, and 1 m~ EDTA. Filters were exposed to Amersham Hype--MP film at -70 "C with an intensilj4ng screen.

RESULTS Tu determine whether gene expression could be detected

after direct injection of DNA into liver, a plasmid with the F? pyrulis luciferase coding sequences under the control of CMV E (pCMvL) was utilized. Following direct injection of 500 pg of pCMvL into five sites in a single lobe of rat liver, a peak of luciferase activity was observed at 24 h. Activity declined rap- idly and was not detected at 7 days. The distribution of trans- fected DNA expression was determined by injecting plasmid DNA containing the gene for p-galactosidase (pCMVp). Histo- chemical analyses of liver sections were performed 48 h after hepatic injection (Fig. lA). Scattered cells with characteristic hepatocyte features, including polygonal cellular morphology, centrally placed uniform nuclei and localization within hepatic plates showed X-gal staining. However, histological analysis also showed acute inflammatory infiltrates near the injection site.

Since transfected gene expression could be adversely affected by inflammation, an attempt was made to supress an inflam- matory reaction by treatment with dexamethasone. Rats and cats were treated with anti-inflammatory doses of dexametha- sone daily, starting 12 h prior to pCMvL injection. Expression of transfected DNA at 48 h was enhanced significantly in the steroid-treated animals (Fig. 2A). Histological analysis of liver

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Dexamethasone Enhancement of Dansfected Gene Expression

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FIG. 2. Dexamethasone enhancement of luciferase protein ex- pression in hepatocytes. A, in uiuo. After direct injection of pCMVL into liver tissue of rats or domestic shorthair cats, luciferase protein

treated (-DEX, solid bars) or treated with dexamethasone (+DEX, accumulation was assayed in single liver lobes at 48 h in animals not

cross-hatched bars). Values are mean 2 S.E., rats (n = 5) and cats (n = 3). *Significantly different from -dexamethasone group, p < 0.01. E , in uitro. After transfection of plasmid DNA into primary rat hepatocytes, transfected cells were either incubated with or without dexamethasone, and luciferase was assayed at 48 h. Transfections were performed with 1 pg of DNA and 7.5 pg of 50:50 dioctadecylamidoglycylspermine: dioleoylphosphatidylethanolamine liposomes in each well of a 24-well microtiter plate (1 x los hepatocytedwell), either with or without 500 nglwell dexamethasone. Six to 10 replicates of each treatment group were performed. Data represent the mean * S.E. of the percentage dexamethasone enhancement found in five separate experiments. *Sig- nificantly different from pRSVL, p < 0.05.

sections from rats injected with pCMvp and treated with dexa- methasone showed an increase in number of transfected hepa- tocytes and less inflammation (Fig. 1 B ) .

To determine if dexamethasone treatment would also en- hance the persistence of gene expression, a time course analy- sis was performed in rats and cats injected with pCMvL and treated with dexamethasone. A substantial initial peak of lu- ciferase activity was observed, followed by a prolonged plateau of expression (Fig. 3). To determine if the effects of dexametha- sone were specific for genes under the control of CMV IE, se- lected experiments were repeated using a plasmid with the

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FIG. 3. Time course of luciferase expression in dexamethasone treated rats (A) or domestic shorthair cats (B) . 500 pg pCMVL diluted in 2-3 ml of Dulbecco's modified Eagle medium were injected into a single lobe of liver in animals treated daily with dexamethasone. Each time point represents the results for one animal terminated, or a single liver lobe removed, and analyzed at the indicated times after intrahepatic injection of the pCMVL.

luciferase cDNA under the control of the RSV LTR (pRSVL) (19). A 9-fold enhancement of luciferase activity with pRSVL was observed in dexamethasone-treated animals compared to untreated controls (459 * 75 pg uersus 51 2 16 pg, mean 2 S.E.) at 48 h. Similar results had been observed with pCMvL, where dexamethasone treatment stimulated a 10-fold increase in lu- ciferase activity (9874 2 2506 pg versus 941 2 190 pg for con- trols, mean 2 S.E.). These results demonstrate that dexametha- sone treatment enhances protein expression from transfected genes with different promoter/enhancer sequences.

To determine if dexamethasone could enhance gene expres- sion in hepatic cells in vitro as well as in vivo, primary rat hepatocytes in culture were transfected with pCMvL or pRSVL. Luciferase activities were increased 7.4 (pCMvL) and 4-fold (pRSVL) in cells treated with dexamethasone compared

29906 Dexamethasone Enhancement of nansfected Gene Expression

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c l a b l l a b l l a b l l a b l l a b l l a b l l a b l

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FIG. 4. The state of injected plasmid DNA in liver tissue over time after direct intrahepatic injection. Southern blot analysis of low molecular weight (extra-chromosomal) DNA after restriction en- zyme digestion with the isoschizomer enzymes NdeII or DpnI. Dupli- cate samples (labeled a and 6) were obtained from livers at the indi- cated times after intrahepatic injection. The first lane is the Southern analysis of a control (labeled c ) with 10,000 copies ofundigested plasmid p c m .

to untreated cells (Fig. 2B). The dexamethasone enhance- ment of luciferase activity in hepatocytes transfected with pCMVL was significantly greater than that seen with pRSVL. These results suggest that dexamethasone treatment may enhance gene expression in vivo solely through transcriptional modulation.

Plasmid DNA, injected into liver tissue, could integrate into the hepatocyte genome, replicate as an episome, persist as a non-replicating episomal plasmid or become fragmented. To investigate these possibilities, both chromosomal and low mo- lecular weight (extra-chromosomal) DNA were isolated a t var- ious times following direct injection of pCMVL and were ana- lyzed for size and methylation patterns. Plasmid DNA was detected in the extra-chromosomal hepatic DNA fractions at all time points (Fig. 4). This plasmid DNA was resistant to NdeII but sensitive to DpnI digestion. Therefore, the injected DNA persisted as a non-replicating episome. To determine if a unique internal luciferase restriction fragment had integrated into chromosomal DNA, the chromosomal DNA fraction was digested and analyzed by Southern blotting. A unique DNA fragment was not detected (data not shown).

DISCUSSION

A variety of hepatic gene delivery techniques have been pro- posed by numerous investigators (1-8). We have found that one of the most reliable and least complex methods is direct injec- tion of plasmid DNA. Our results extend previous observations for direct DNA injection in skeletal and cardiac muscle (11, 12) and suggest that uptake of injected DNA may represent a gen- eral property of many tissues. The lack of hepatocyte transfec- tion following direct DNA injection reported by other research- ers (16-18) may have been due to a different injection technique. Also, dexamethasone treatment enhanced and pro- longed transfected gene activity in this study, probably by di- rect transcriptional activation and possibly by reducing inflam- mation at the injection site.

Cultured primary hepatocytes, transfected with either pC- MVL or pRSVL, responded to dexamethasone treatment with a marked increase in luciferase activity compared to untreated

controls. Consistent with previous studies of transfected pri- mary hepatocytes, CMV IE promoted more efficient gene ex- pression than the RSV LTR (6). In addition, pCMVL trans- fected cells showed a greater enhancement of luciferase activity in response to dexamethasone than those transfected with pRSVL. The transcriptional elements of the RSV LTR and CMV IE are not known to respond to glucocorticoids. However, CMV IE contains repeated sequences with glucocorticoid response element (GRE) homology. The 6-base sequence AGTACA is found four times within the CMV IE (beginning a t base 148,

TACA TCT ACGTAT; and 312, AGTACA TCA ATGGGC) (24). Two of these potential 5' GRE-like elements are similar to the 5' consensus GRE 5"GGTACA NNN TGTTCT-3', but vary sig- nificantly from the 3' GRE sequence consensus (25). Repeated GRE-like sequences within the CMV IE, and increased expres- sion of pCMVL after dexamethasone treatment of cultured hepatocytes provide evidence for dexamethasone/glucocorticoid receptor activation of CMV IE-driven transcription. Interest- ingly, treatment of MRC-5 cells with dexamethasone enhances the growth and cytopathicity of isolates of CMV from clinical specimens (26, 27). Viral recovery from MRC-5 cells is en- hanced by pretreatment with dexamethasone for 24 h and is associated with increased CMV IE antigen expression. Cordycepin, an inhibitor of mRNA maturation, blocks this ef- fect. The enhancement by pre-treatment with dexamethasone can be interpreted as indirect dexamethasone induction of CMV IE, perhaps by inducing synthesis of a cellular transcrip- tion factor which then interacts with CMV IE regulatory ele- ments (27). Further analysis of the interactions of dexametha- sone and CMV IE elements will be needed to clarify the mechanism(s) of glucocorticoid action.

Dexamethasone-mediated CMV IE activation may also con- tribute to the increased incidence of clinical CMV disease in transplant recipients and AIDS patients treated with cortico- steroids. Corticosteroid immunosuppression results in clinical CMV infection of 60% of CMV-seronegative and 90% of CMV- seropositive renal transplant patients. In human immunodefi- ciency virus-infected individuals, corticosteroid treatment has been associated with CMV pneumonitis and other forms of clinical CMV disease (28). The temporal regulation of CMV replication is absolutely dependent on immediate early gene expression. Our results support the hypothesis that steroid activation of CMV IE transcription enhances viral replication and increases CMV viremia in corticosteroid-treated patients.

In vivo transduction of hepatocytes (or in vitro transduction followed by hepatocyte infusion) using retrovirus or herpesvi- rus vectors containing the CMV IE is associated with a rapid loss of CMV IE activity (3, 29). However, inactivation of the CMV IE has not been observed in cultured hepatocytes (3). The in vivo loss of protein expression from virally transduced CMV IE sequences has been attributed to promoter inactivation, which may be a consequence of associated viral sequences. The direct in vivo delivery of pCMVL has allowed us to test CMV IE inactivation in the absence of other viral sequences. These re- sults indicate that the promoter inactivation of CMV IE previ- ously reported (3, 29) occurs without other viral sequences but can be substantially reduced by treatment with glucocorticoids.

Gene augmentation therapy has the potential for insertional mutagenesis following random integration of transfected se- quences. Activation of adjacent oncogenes has occurred follow- ing retroviral integration (10). Persistent gene expression from a non-replicating plasmid without detectable integration was first reported following direct injection of skeletal muscle (11). Similar results have been observed with targeted hepatic transfection followed by partial hepatectomy (8). However, per-

AGTACA TCAAGTGTA; 228, AGTACA TGA CCTTAT 261, AG-

Dexamethasone Enhancement of fiansfected Gene Expression 29907

sistent gene expression may represent transcription from un- detected integrated DNA (11). The finding of persistent unin- tegrated DNA with a bacterial methylation pattern following intrahepatic injection supports the former hypothesis but does not exclude the latter.

These studies indicate that striated muscle cells are not unique in their capability to take up and express plasmid DNA after direct injection. Hepatocytes, and probably many other cell types, are transfected readily by direct DNA injection into tissue. Enhancement of transfected gene expression in vivo after treatment with dexamethasone demonstrates that phar- macologically defined polynucleotide delivery systems will be useful for analyses of the in vivo interactions of genes and organisms, without the cost and technical demands of more complex methods such as constructing germ line transgenic animals.

Acknowledgments-We thank Karl Mack for the kind gift of pCMvL, and Suresh Subramani for pRSVL. We also thank Peter Bany and Paul Luciw for helpful discussions about CMV and viral latency, and Philip Schneider for assistance with the initial rat surgery.

REFERENCES

2. Hatzoglou, M., Lamers, W., Bosch, F., Wynshaw-Boris, A,, Clapp, D. W., and 1. Kaleko, M., Garcia, J. V., and Miller, A. D. (1991) Hum. Gene Therapy 2,2732

Hanson, R. W. (1990) J. Bwl. Chem. 265, 17285-17293 3. Kay, M. A., Baley, P., Rothenberg, S., Leland, F., Fleming, L., Ponder, K. P., Liu,

T., Finegold, M., Darlington, G., and Pokorny, W. (1992) Proc. Natl. Acad.

4. Nicolau, C., and Cudd,A. (1989) Crit. Reu. Then Drug Carrier Syst. 6,239-271 Sei. U. 5. A. 89, 89-93

5. Leibiger, I., Leibiger, B., Sarrach, D., Walther, R., and Zuhlke, H. (1990)

6. Ponder, K. P., Dunbar, R. P., Wilson, D. R., Darlington, G. J., and Woo, S. L. Biomed. Biochim. Acta 49, 1193-1200

7. wu

9. Donahue, R. E., (1992) J. Exp. Med. 176, 1125-1135 Biol. Chem. 267, 11483-11489

10. Nusse, R., Theunissen, H., Wagenaar, E., Rijsewijk, F., Gennissen,A., Otte, A.,

11. Wolff, J. A,, Malone, R. W., Williams, P., Chong, W., Acsadi, G., Jani, A,, and

12. Lin, H., Parmacek, M. S., Morle, G., Bolling, S., and Leiden, J. M. (1990)

13. Will, H., Cattaneo, R., Koch, H. G., Darai, G., Schaller, H., Schellekens, H., van

14. Seeger, C., Marion, P. L., Ganem, D., and Varmus, H. E (1984) Proc. Natl. Acad.

15. Dubensky, T. W., Campbell, B. A., and Villarreal, L. P. (1984) Proc. Natl. Acad.

16. Wolff, J. A., Williams, P., Acsadi, G., Jiao, S., Jani, A,, and Chong, W. (1991)

17. Acsadi, G., Jiao, S. S., Jani, A,, Duke, D., Williams, P., Chong, W., and Wolff, J.

18. Jiao, S., Williams, P., Berg, R. K., Hodgeman, B. A,, Liu, L., Repetto, G., and

19. de Wet, J. R., Wood, K. V., DeLuca, M., Helinski, D. R., and Subramani, S.

20. Price, J. (1987) Proc. Natl. Acad. Sci. U. 5. A. 84, 156-160 21. Berry M. N., and Friend, D. S. (1969) J. Cell Biol. 43, 506-520 22. Hirt, B. J. (1967) J. Mol. Biol. 26, 365-369 23. Church, G. M., and Gilbert, W. (1984) Proc. Natl. Acad. Sci. U. S . A . 81,

24. Akrigg, A., Wilkinson, G. W., and Oram, J. D. (1985) Virus Res. 2, 107-121 25. Evans, R. M. (1988) Science 240,889-895 26. Tanaka, J., Ogura, T., Kamiya, S., Yoshie, T., Yabuki, Y., and Hatano, M. (1984)

27. West, P. G.,Aldrich, B., Hartwig, R., and Haller, G. J. (1988) J. Clin. Microbial.

Schuuring, E., and van Ooyen, A. (1990) Mol. Cell. Biol. 10,41704179

Felgner, P. L. (1990) Science 247, 1465-1468

Circulation 82, 2217-2221

Eerd, P. M., and Deinhardt, F. (1982) Nature 299,740-742

Sci. U. 5. A. 81, 5849-5852

Sci. U. S . A. 81, 7529-7533

BwTechniques 11,474485

A. (1991) New Biol. 3, 71-81

Wolff, J. A. (1992) Hum. Gene Therapy 3,2143

(1987) Mol. Cell. Biol. 7, 725-737

1991-1995

Virology 136, 448-452

26. 2.510-2.514 28. Nelson, M. R., Erskine, D., Hawkins, D. A., and Gazzard, B. G. (1993) AIDS

,"" ""

(Phila.) 7. 375-378 29. Miyanohara, A., Johnson P. A., Elam, R. L., Dai, Y., Witztum, J. L., Verma, I.

. ,

M., and Friedmann, T. (1992) New Biol. 4,238-246