Encoding Enhanced Green Fluorescent Protein Expression Research Articles

Identification of Novel Antiviral Compounds Targeting Entry of Hantaviruses.

Hemorrhagic fever viruses, among them orthohantaviruses, arenaviruses and filoviruses, are responsible for some of the most severe human diseases and represent a serious challenge for public health. The current limited therapeutic options and available vaccines make the development of novel efficacious antiviral agents an urgent need. Inhibiting viral attachment and entry is a promising strategy for the development of new treatments and to prevent all subsequent steps in virus infection. Here, we developed a fluorescence-based screening assay for the identification of new antivirals against hemorrhagic fever virus entry. We screened a phytochemical library containing 320 natural compounds using a validated VSV pseudotype platform bearing the glycoprotein of the virus of interest and encoding enhanced green fluorescent protein (EGFP). EGFP expression allows the quantitative detection of infection and the identification of compounds affecting viral entry. We identified several hits against four pseudoviruses for the orthohantaviruses Hantaan (HTNV) and Andes (ANDV), the filovirus Ebola (EBOV) and the arenavirus Lassa (LASV). Two selected inhibitors, emetine dihydrochloride and tetrandrine, were validated with infectious pathogenic HTNV in a BSL-3 laboratory. This study provides potential therapeutics against emerging virus infection, and highlights the importance of drug repurposing.

Avian Adeno-Associated Viral Transduction of the Postembryonic Chicken Retina.

PurposeThe posthatching chicken is a valuable animal model for research, but molecular tools needed for altering its gene expression are not yet available. Our purpose here was to adapt the adeno-associated viral (AAV) vector method, used widely in mammalian studies, for use in investigations of the chicken retina. We hypothesized that the recently characterized avian AAV (A3V) vector could effectively transduce chick retinal cells for manipulation of gene expression, after intravitreal or subretinal injection.MethodsA3V encoding enhanced green fluorescent protein (EGFP) was injected intravitreally or subretinally into P1-3 chick eye and left for 7 to 10 days. Retinas were then sectioned or flat-mounted and visualized via laser-scanning confocal microscopy for analysis of expression and imaging of retinal cells.ResultsIntravitreal A3V-EGFP injection resulted in EGFP expression in a small percent of retinal cells, primarily those with processes and/or cell bodies near the vitreal surface. In contrast, subretinal injection of A3V-EGFP within confined retinal “blebs” produced high rates of transduction of rods and all types of cones. Some examples of all other major retinal cell types, including horizontal, amacrine, bipolar, ganglion, and Müller cells, were also transduced, although with much lower frequency than photoreceptors.ConclusionsA3V is a promising tool for investigating chick retinal cells and circuitry in situ. This novel vector can be used for studies in which local photoreceptor transduction is sufficient for meaningful observations.Translational RelevanceWith this vector, the postembryonic chick retina can now be used for preclinical trials of gene therapy for prevention and treatment of human retinal disease.

Targeted gene delivery to the synovial pannus in antigen-induced arthritis by ultrasound-targeted microbubble destruction in vivo

The purpose of this study was to optimize an ultrasound-targeted microbubble destruction (UTMD) technique to improve the in vivo transfection efficiency of the gene encoding enhanced green fluorescent protein (EGFP) in the synovial pannus in an antigen-induced arthritis rabbit model. A mixture of microbubbles and plasmids was locally injected into the knee joints of an antigen-induced arthritis (AIA) rabbits. The plasmid concentrations and ultrasound conditions were varied in the experiments. We also tested local articular and intravenous injections. The rabbits were divided into five groups: (1) ultrasound+microbubbles+plasmid; (2) ultrasound+plasmid; (3) microbubble+plasmid; (4) plasmid only; (5) untreated controls. EGFP expression was observed by fluorescent microscope and immunohistochemical staining in the synovial pannus of each group. The optimal plasmid dosage and ultrasound parameter were determined based on the results of EGFP expression and the present and absent of tissue damage under light microscopy. The irradiation procedure was performed to observe the duration of the EGFP expression in the synovial pannus and other tissues and organs, as well as the damage to the normal cells. The optimal condition was determined to be a 1-MHz ultrasound pulse applied for 5min with a power output of 2W/cm2 and a 20% duty cycle along with 300μg of plasmid. Under these conditions, the synovial pannus showed significant EGFP expression without significant damage to the surrounding normal tissue. The EGFP expression induced by the local intra-articular injection was significantly more increased than that induced by the intravenous injection. The EGFP expression in the synovial pannus of the ultrasound+microbubbles+plasmid group was significantly higher than that of the other four groups (P<0.05). The expression peaked on day 5, remained detectable on day 40 and disappeared on day 60. No EGFP expression was detected in the other tissues and organs. The UTMD technique can significantly enhance the in vivo gene transfection efficiency without significant tissue damage in the synovial pannus of an AIA model. Thus, this could become a safe and effective non-viral gene transfection procedure for arthritis therapy.

Development of human cell biosensor system for genotoxicity detection based on DNA damage-induced gene expression

BackgroundHuman exposure to genotoxic agents in the environment and everyday life represents a serious health threat. Fast and reliable assessment of genotoxicity of chemicals is of main importance in the fields of new chemicals and drug development as well as in environmental monitoring. The tumor suppressor gene p21, the major downstream target gene of activated p53 which is responsible for cell cycle arrest following DNA damage, has been shown to be specifically up-regulated by genotoxic carcinogens. The aim of our study was to develop a human cell-based biosensor system for simple and fast detection of genotoxic agents.MethodsMetabolically active HepG2 human hepatoma cells were transfected with plasmid encoding Enhanced Green Fluorescent Protein (EGFP) under the control of the p21 promoter (p21HepG2GFP). DNA damage was induced by genotoxic agents with known mechanisms of action. The increase in fluorescence intensity, due to p21 mediated EGFP expression, was measured with a fluorescence microplate reader. The viability of treated cells was determined by the colorimetric MTS assay.ResultsThe directly acting alkylating agent methylmethane sulphonate (MMS) showed significant increase in EGFP production after 48 h at 20 μg/mL. The indirectly acting carcinogen benzo(a)pyren (BaP) and the cross-linking agent cisplatin (CisPt) induced a dose- dependent increase in EGFP fluorescence, which was already significant at concentrations 0.13 μg/mL and 0.41 μg/mL, respectively. Vinblastine (VLB), a spindle poison that does not induce direct DNA damage, induced only a small increase in EGFP fluorescence intensity after 24 h at the lowest concentration (0.1 μg/mL), while exposure to higher concentrations was associated with significantly reduced cell viability.ConclusionsThe results of our study demonstrated that this novel assay based on the stably transformed cell line p21HepG2GFP can be used as a fast and simple biosensor system for detection of genetic damage caused by chemical agents.

FLUORESCENCE ACTIVATED CELL SORTING OF TRANSIENTLY TRANSFECTED As4.1 CELLS SHOWS RENIN ENHANCER DIRECTS ON/OFF SWITCHING OF RENIN PROMOTER IN VITRO

1. The proximal promoter of the renin gene is weak and its activity is influenced by a strong, far-upstream enhancer. This and the ability of renin expression in renal afferent arteriolar cells to be 'recruited' under chronic stimulation is consistent with the on/off switching (variegation) model of gene expression. If true, this would provide an example in which variegation controls a physiologically regulable gene. 2. The present study tested the hypothesis that renin promoter activity may accord with the variegation model, at least in individual juxtaglomerular (mouse As4.1) cells in vitro. 3. As4.1 cells were transiently transfected with constructs containing the mouse renin (Ren-1c) enhancer adjacent to the Ren-1c promoter and a linked reporter gene encoding enhanced green fluorescent protein (EGFP). The EGFP signal from individual cells was monitored by fluorescence activated cell sorting. 4. In the presence of the renin enhancer there was 10-fold higher EGFP expression in transfected cells compared with cells transfected with EGFP constructs containing the promoter alone. There was, moreover, an 8-fold increase in the number of EGFP expressing cells. However, EGFP expression in individual transfected cells was similar in the presence or absence of the enhancer. 5. Results from the in vitro system used suggest that the Ren-1c enhancer does not regulate the rate of promoter activity, but rather increases the probability of achieving an active transcriptional state. Limitations of these findings are discussed.

Use of microarrays in transfection of mammalian cells with dicer-digested small interfering RNAs

Transfection cell arrays provide promising methods for the high-throughput analysis of gene functions. Such analysis is performed efficiently by using small interfering RNA (siRNA). To extend the usefulness of microarrays, this study was devoted to implementing siRNA prepared from complementary DNA (cDNA). The preparation of siRNA involves the transcription of cDNA to generate double-stranded RNA (dsRNA) followed by digestion with endoribonuclease, dicer, by which one can obtain the library of siRNAs without target sequence optimization. In this study, endoribonuclease-digested siRNA (d-siRNA) was prepared using cDNA encoding enhanced green fluorescent protein (EGFP) and loaded onto micropatterned substrates through electrostatic interactions of siRNA complex with substrates. We observed that d-siRNA loading was comparable to the case with siRNA prepared by chemical synthesis. When cotransfected with EGFP plasmid into human embryonic kidney 293 (HEK293) cells on a microarray, d-siRNA suppressed EGFP expression in a loading-dependent manner. In addition, d-siRNA triggered gene silencing at a level similar to that of chemically synthesized siRNA. The similarity between the two types of siRNA regarding silencing efficiency suggests that heterogeneity in nucleotide sequences of d-siRNA has minor effects. From these results, we conclude that the combination of d-siRNA with the array technology provides useful tools for high-throughput screening of gene functions.

Disruption of E-Cadherin-Mediated Adhesion Induces a Functionally Distinct Pathway of Dendritic Cell Maturation

The maturation of dendritic cells (DCs) after exposure to microbial products or inflammatory mediators plays a critical role in initiating the immune response. We found that maturation can also occur under steady-state conditions, triggered by alterations in E-cadherin-mediated DC-DC adhesion. Selective disruption of these interactions induced the typical features of DC maturation including the upregulation of costimulatory molecules, MHC class II, and chemokine receptors. These events were triggered at least in part by activation of the beta-catenin pathway. However, unlike maturation induced by microbial products, E-cadherin-stimulated DCs failed to release immunostimulatory cytokines, exhibiting an entirely different transcriptional profile. As a result, E-cadherin-stimulated DCs elicited an entirely different T cell response in vivo, generating T cells with a regulatory as opposed to an effector phenotype. These DCs induced tolerance in vivo and may thus contribute to the elusive steady-state "tolerogenic DCs."

Translational initiation in Leishmania tarentolae and Phytomonas serpens (Kinetoplastida) is strongly influenced by pre-ATG triplet and its 5′ sequence context

To investigate the influence of sequence context of translation initiation codon on translation efficiency in Kinetoplastida, we constructed a library of expression plasmids randomized in the three nucleotides prefacing ATG of a reporter gene encoding enhanced green fluorescent protein (EGFP). All 64 possible combinations of pre-ATG triplets were individually stably integrated into the rDNA locus of Leishmania tarentolae and the resulting cell lines were assessed for EGFP expression. The expression levels were quantified directly by measuring the fluorescence of EGFP protein in living cells and confirmed by Western blotting. We observed a strong influence of the pre-ATG triplet on the level of protein expression over a 20-fold range. To understand the degree of evolutionary conservation of the observed effect, we transformed Phytomonas serpens, a trypanosomatid parasite of plants, with a subset of the constructs. The pattern of translational efficiency mediated by individual pre-ATG triplets in this species was similar to that observed in L. tarentolae. However, the pattern of translational efficiency of two other proteins (red fluorescent protein and tetracycline repressor) containing selected pre-ATG triplets did not correlate with either EGFP or each other. Thus, we conclude that a conserved mechanism of translation initiation site selection exists in kinetoplastids that is strongly influenced not only by the pre-ATG sequences but also by the coding region of the gene.

Incorporation of quantum dots on virus in polycationic solution

Developing methods to label viruses with fluorescent moieties has its merits in elucidating viral infection mechanisms and exploring novel antiviral therapeutics. Fluorescent quantum dots (QDs), an emerging probe for biological imaging and medical diagnostics, were employed in this study to tag retrovirus encoding enhanced green fluorescent protein (EGFP) genes. Electrostatic repulsion forces generated from both negatively charged retrovirus and QDs were neutralized by cationic Polybrene, forming colloidal complexes of QDs-virus. By examining the level of EGFP expression in 3T3 fibroblast cells treated with QDs-tagged retroviruses for 24 hours, the infectivity of retrovirus incorporated with QDs was shown to be only slightly decreased. Moreover, the imaging of QDs can be detected in the cellular milieu. In summary, the mild method developed here makes QDs-tagged virus a potential imaging probe for direct tracking the infection process and monitoring distribution of viral particles in infected cells.

655. Compacted DNA Nanoparticles Effectively Transfect Brain Cells In Vitro and In Vivo

This study determined the feasibility of using DNA compacted into unimolecular nanoparticles as a gene therapy delivery system for post-mitotic brain cells. DNA compacted with polyethylene glycol (PEG)-substituted 30-mer lysine peptides has been shown to effectively transfect multiple types of non-dividing cells and tissues, including growth-arrested neuroblastoma cells, and may be a viable system for the delivery of neurotrophic factors, such as glial cell line-derived neurotrophic factor (GDNF), to dopaminergic neurons. This formulation of DNA nanoparticles is non-inflammatory and non-immunogenic in peripheral organs, and therefore may circumvent the problem of immunogenicity that is inherent with some viral vectors. In this study we examined whether specific nanoparticle formulations of single DNA molecules encoding enhanced green fluorescent protein (EGFP) are capable of transfecting brain cells in primary ventral midbrain cultures or after direct injection into the rat striatum. Ellipsoidal and rod-like formulations of DNA nanoparticles were prepared by using lysine peptides having either trifluoroacetate (TFA) or acetate, respectively, as the polycation counterion at the time of DNA mixing. Primary ventral midbrain cultures were maintained for 5 days. During the first 4 days, cultures were exposed to DNA compacted into the acetate rod nanoparticles [250 ng/well; 5.0 |[times]| 1010 particles]; fresh media containing nanoparticles was added to the cultures on a daily basis. On the fifth day in vitro (DIV 5), |[sim]|99% of the cells expressing either the neural marker NeuN or the glial marker glial fibrillary acidic protein (GFAP) were also EGFP+; similarly, most tyrosine hydroxylase-positive cells also co-expressed EGFP. In a second study, ellipsoidal and rod-like nanoparticles were injected directly into the striatum of young adult rats and 4-5 days later we observed EGFP expression in |[sim]|50% of both neurons and glia located in the brain tissue surrounding the injection site. There was no evidence of an inflammatory cell infiltrate at the injection site. In conclusion, the results from these in vitro and in vivo studies suggest that compacted DNA nanoparticles can be used as a non-viral gene therapy technique to efficiently transfect post-mitotic brain cells and induce transgene expression in both neurons and glia. This study was supported by NS42862 (NINDS/NIH) and DAMD 17-01-1-0786 (DOD).

Efficient gene transfer into murine embryonic stem cells by nucleofection

Genetic manipulation of embryonic stem (ES) cells is performed by non-viral as well as viral transfection methods. We tested the recently developed nucleofection method delivering plasmid DNA directly into the nucleus for the introduction of a plasmid encoding enhanced green fluorescent protein (EGFP) into murine ES cells. Cell viability decreased from 77% before to 40% 24 h after nucleofection. Transfection effciencies in viable stem cells were between 85% and 96% with high levels of EGFP expression [mean fluorescence intensity (MFI): 630 +/- 90] 24 h after nucleofection. After a two week culture in geneticin (G418) selection medium, nearly 50% of the stem cells were EGFP positive and continued transgene expression (MFIs: 120-240) for a two further weeks. We conclude that nucleofection is an efficient nonviral gene transfer method for the introduction of genes into murine ES cells.

Stable transgene expression in tumors and metastases after transduction with lentiviral vectors based on human immunodeficiency virus type 1.

The relatively low efficiency of target cell transduction and variations in the stability of transgene expression by retroviral vectors based on the Moloney murine leukemia virus (MoMLV) are major impediments to the use of such vectors in cancer gene therapy approaches. The present study was designed to investigate the stability and efficiency of transgene expression in human lung and breast cancer cell lines transduced with vectors based on human immunodeficiency virus type 1 (HIV-1) in vitro and in vivo in nude mouse models of metastasis. H460 lung carcinoma cells and MDA-MB-231 breast carcinoma cells were transduced with lentiviral vectors encoding enhanced green fluorescent protein (EGFP) and beta-galactosidase (beta-Gal), respectively. Transduced H460 cells were administered to nude mice by either intravenous or subcutaneous injection and MDA-MB-231 cells were implanted orthotopically into the mammary fat pad of such mice to induce primary tumor and metastatic lung tumor formation. High-level EGFP expression was maintained in transduced H460 cells in metastatic lung nodules for up to 6 weeks and transgene expression in vitro persisted for at least 23 days after retrieval of EGFP-positive H460 cells from the lungs of tumor-bearing mice and subsequent cultivation in vitro. Likewise, beta-Gal expression levels in metastatic MDA-MB-231 cells in lungs remained high for up to 11 weeks. Southern blot analyses carried out with DNA from lung nodules showed that proviral DNAs in H460 cells were maintained stably over many cell generations and during subsequent reimplantation in vivo. However, molecular analyses revealed variations in transgene copy numbers and expression levels among individual lung clones. These results demonstrate the usefulness of HIV-1-based lentiviral vectors for sustained and stable transgene expression in human lung and breast cancer cell lines in vitro and in vivo.

Indirect intracoronary delivery of adenovirus encoding adenylyl cyclase increases left ventricular contractile function in mice.

We performed indirect intracoronary delivery of adenovirus vectors in mice and explored techniques including hypothermia and pharmacological means to increase cardiac gene transfer. Mice were maintained in a normothermic state or cooled to 25 degrees C. The aorta or both the pulmonary artery and aorta were clamped while a needle was advanced into the left ventricular cavity to deliver adenovirus vectors encoding enhanced green fluorescent protein (EGFP) or murine adenylyl cyclase type VI (AC(VI)) with saline, sodium nitroprusside, acetylcholine, or serotonin. Clamping was maintained for 30 s (normothermia) or 2 min (25 degrees C) after adenovirus administration. Mice were killed 7 or 21 days later, and hearts were examined for EGFP expression. Compared with clamping the aorta alone and with no cooling, gene transfer was increased as follows: 1) 1.3-fold with hypothermia to extend dwell time; 2) 4.5-fold by clamping the aorta and the pulmonary artery; 3) 11.4-fold with nitroprusside administration; 4) 11.8-fold with serotonin addition, and 5) 14.3-fold with acetylcholine delivery. Gene expression remained substantial at 21 days, and no significant inflammatory response was seen. Efficacy of the method was tested by performing gene transfer of adenovirus encoding AC(VI). Fourteen days after gene transfer, hearts isolated from mice that received adenovirus encoding AC(VI) showed increased contractile function. Indirect intracoronary delivery of adenovirus vectors in mice is associated with efficient cardiac gene transfer and increased left ventricular function after AC(VI) gene transfer.

Effects of human locus control region elements HS2 and HS3 on human β-globin gene expression in transgenic mouse

The locus control region (LCR) is the most important cis -element in the regulation of β-globin gene expression. DNaseI-hypersensitive site (HS) 2 and HS3 are two significant components of β-LCR. To examine the effect of HS2, HS3, and HS2–HS3 (combination of HS2 and HS3) on the spatial and temporal expression of the human β-globin gene, we have produced transgenic mice with constructs, in which the gene encoding enhanced green fluorescent protein (EGFP) is driven by β-globin promoter and under the control of HS2, HS3, and HS2–HS3, respectively. The results showed that HS2 and HS3 each had the same enhancement activity in regulation of β-globin gene expression in transgenic mice. When HS2 and HS3 were in combination (HS2–HS3), the two cis -elements showed a marked synergy in regulating β-globin gene spatial and temporal expression as well as its expression level in transgenic mice although the EGFP expression varied largely among different transgenic mouse litters. The results also showed that HS2 was able to confer β-globin gene expression in embryonic yolk sac, fetal liver, and adult bone marrow, which was not developmentally stage-specific, while HS3 could confer the same β-globin gene expression in the adult. Thus, HS3 was different from HS2, the former being more important for specific expression of β-globin gene in the developmental stages and the switch of γ→β-globin genes. Our results indicate that the mechanism of γ→β switch could be best explained by the “divided model.”

Effects of human locus control region elements HS2 and HS3 on human β-globin gene expression in transgenic mouse

The locus control region (LCR) is the most important cis-element in the regulation of β-globin gene expression. DNaseI-hypersensitive site (HS) 2 and HS3 are two significant components of β-LCR. To examine the effect of HS2, HS3, and HS2–HS3 (combination of HS2 and HS3) on the spatial and temporal expression of the human β-globin gene, we have produced transgenic mice with constructs, in which the gene encoding enhanced green fluorescent protein (EGFP) is driven by β-globin promoter and under the control of HS2, HS3, and HS2–HS3, respectively. The results showed that HS2 and HS3 each had the same enhancement activity in regulation of β-globin gene expression in transgenic mice. When HS2 and HS3 were in combination (HS2–HS3), the two cis-elements showed a marked synergy in regulating β-globin gene spatial and temporal expression as well as its expression level in transgenic mice although the EGFP expression varied largely among different transgenic mouse litters. The results also showed that HS2 was able to confer β-globin gene expression in embryonic yolk sac, fetal liver, and adult bone marrow, which was not developmentally stage-specific, while HS3 could confer the same β-globin gene expression in the adult. Thus, HS3 was different from HS2, the former being more important for specific expression of β-globin gene in the developmental stages and the switch of γ→β-globin genes. Our results indicate that the mechanism of γ→β switch could be best explained by the “divided model.”

Lentivirus vector-mediated hematopoietic stem cell gene transfer of common gamma-chain cytokine receptor in rhesus macaques.

Nonhuman primate model systems of autologous CD34+ cell transplant are the most effective means to assess the safety and capabilities of lentivirus vectors. Toward this end, we tested the efficiency of marking, gene expression, and transplant of bone marrow and peripheral blood CD34+ cells using a self-inactivating lentivirus vector (CS-Rh-MLV-E) bearing an internal murine leukemia virus long terminal repeat derived from a murine retrovirus adapted to replicate in rhesus macaques. In vitro cytokine stimulation was not required to achieve efficient transduction of CD34+ cells resulting in marking and gene expression of the reporter gene encoding enhanced green fluorescent protein (EGFP) following transplant of the CD34+ cells. Monkeys transplanted with mobilized peripheral blood CD34+ cells resulted in EGFP expression in 1 to 10% of multilineage peripheral blood cells, including red blood cells and platelets, stable for 15 months to date. The relative level of gene expression utilizing this vector is 2- to 10-fold greater than that utilizing a non-self-inactivating lentivirus vector bearing the cytomegalovirus immediate-early promoter. In contrast, in animals transplanted with autologous bone marrow CD34+ cells, multilineage EGFP expression was evident initially but diminished over time. We further tested our lentivirus vector system by demonstrating gene transfer of the human common gamma-chain cytokine receptor gene (gamma(c)), deficient in X-linked SCID patients and recently successfully used to treat disease. Marking was 0.42 and.001 HIV-1 vector DNA copy per 100 cells in two animals. To date, all EGFP- and gamma(c)-transplanted animals are healthy. This system may prove useful for expression of therapeutic genes in human hematopoietic cells.

Retrovirus-mediated transfer of the herpes simplex virus thymidine kinase and enhanced green fluorescence protein genes in primary T lymphocytes.

The EGFP-tk retroviral vector, encoding enhanced green fluorescent protein (EGFP) and the herpes simplex virus thymidine kinase (HSV-tk) packaged in a Phoenix amphotropic cell line, was used to transduce healthy donor T lymphocytes. Infection yielded a mean of 41.8 +/- 9.3% SD (range 31.1-48.4%) EGFP-positive cells and a mean of 92 +/- 2% SD (range 90-94%) after cell sorting. EGFP expression remained stable for 30 d after infection. The entire gene transfer procedure had no significant effect on lymphocyte subsets and slightly reduced clonogenicity. Ganciclovir (gcv) treatment (1 microg/ml x 10 d) killed all EGFP-positive cells in the transduced and transduced/sorted populations, but had no effect on untransduced controls. Our results show that primary T lymphocytes can be transduced using an EGFP-tk vector that yields a homogeneous infected population without affecting lymphocyte subsets, function and clonogenicity.

In Situ Demonstration of Inhibitory Effects of Hammerhead Ribozymes That Are Targeted to the Hepatitis Bx Sequence in Cultured Cells

Chronic hepatitis B virus (HBV) infection is endemic to several populous areas of the world and is frequently complicated by hepatocellular carcinoma. Ribozymes can be designed to cleave target RNA sequences specifically and show promise for the treatment of HBV infection. Demonstration of intracellular inhibition of HBV gene expression, essential to developing therapeutic ribozymes, has been the aim of this investigation. We generated two vectors encoding hammerhead ribozymes that target the HBx region of HBV. Plasmids containing intact HBV sequences or a modification in which the preS2/S region was replaced by DNA encoding enhanced green fluorescent protein (EGFP) were used to test ribozyme action in transfected cells. Both ribozymes inhibited surface antigen secretion and EGFP expression similarly. The measurement of EGFP expression is convenient to assess ribozyme action in situ and effective targeting of HBV sequences that are common to all HBV transcripts is potentially useful to develop strategies to counter HBV infection.