Direct DNA Injection Research Articles

METHODS OF GENETIC TRANSFORMATION: MAJOR EMPHASIS TO CROP PLANTS

Advancements in gene transfer technology have indeed opened up exciting possibilities for more effectively manipulating the genetic makeup of live organisms, ranging from microorganisms to plants and animals. Direct and indirect transformations are the two basic types of gene transfer techniques. Indirect method comprises Agrobacterium mediated method as it involves intermediate host between gene of interest and target and this method is most opted one out of all present. Direct gene transformation methods, on the other hand, do not involve the use of an intermediate host organism. Instead, they rely on physical means to transfer genes between cells. Biolistic transformation uses high-velocity particles to deliver DNA into target cells, while microinjection and macroinjection involve the direct injection of DNA into cells. Protoplast fusion combines the genetic material of two different cells by fusing their protoplasts. Natural methods for gene transfer encompass mechanisms that occur naturally in various organism, includes transposition, conjugation, phage and retroviral transductions and bacterial transformation. Chemical techniques utilize chemical agents to facilitate gene transfer, such as calcium phosphate-mediated transformation, polyethylene glycol (PEG)-mediated transformation, DEAE (Diethylethanolamine)-Dextran-mediated transformation. Genes can be also being transferred using electrical techniques such as electroporation and electrofusion. Crop improvement and trait improvement are now being hastened by the fast-rising number of sequenced plant genomes, information from functional genomics data to understand gene function, innovative gene cloning, and tissue culture techniques. Despite being indispensable, its progress is still hindered by the fact that many plant species and agricultural genotypes exhibit low transformability or are resistant to established tissue culture and regeneration conditions. Here, we review the techniques employed in plant transformation and provide a concise overview of their evolution in agricultural crops, from their first inception to present time.

The Dual Role of the Innate Immune System in the Effectiveness of mRNA Therapeutics.

Advances in molecular biology have revolutionized the use of messenger RNA (mRNA) as a therapeutic. The concept of nucleic acid therapy with mRNA originated in 1990 when Wolff et al. reported successful expression of proteins in target organs by direct injection of either plasmid DNA or mRNA. It took decades to bring the transfection efficiency of mRNA closer to that of DNA. The next few decades were dedicated to turning in vitro-transcribed (IVT) mRNA from a promising delivery tool for gene therapy into a full-blown therapeutic modality, which changed the biotech market rapidly. Hundreds of clinical trials are currently underway using mRNA for prophylaxis and therapy of infectious diseases and cancers, in regenerative medicine, and genome editing. The potential of IVT mRNA to induce an innate immune response favors its use for vaccination and immunotherapy. Nonetheless, in non-immunotherapy applications, the intrinsic immunostimulatory activity of mRNA directly hinders the desired therapeutic effect since it can seriously impair the target protein expression. Targeting the same innate immune factors can increase the effectiveness of mRNA therapeutics for some indications and decrease it for others, and vice versa. The review aims to present the innate immunity-related 'barriers' or 'springboards' that may affect the development of immunotherapies and non-immunotherapy applications of mRNA medicines.

Sonodelivery in Skeletal Muscle: Current Approaches and Future Potential.

There are currently multiple approaches to facilitate gene therapy via intramuscular gene delivery, such as electroporation, viral delivery, or direct DNA injection with or without polymeric carriers. Each of these methods has benefits, but each method also has shortcomings preventing it from being established as the ideal technique. A promising method, ultrasound-mediated gene delivery (or sonodelivery) is inexpensive, widely available, reusable, minimally invasive, and safe. Hurdles to utilizing sonodelivery include choosing from a large variety of conditions, which are often dependent on the equipment and/or research group, and moderate transfection efficiencies when compared to some other gene delivery methods. In this review, we provide a comprehensive look at the breadth of sonodelivery techniques for intramuscular gene delivery and suggest future directions for this continuously evolving field.

New microRNA biotechnology as an in vivo therapeutic molecule to inhibit cancer

MicroRNAs (miRs) are biomarkers of cancer development and progression, they regulate cell fate, stemness and embryonic development. It is well-known that miRs play a role in cancer and are associated with numerous types of cancer as either oncogenes or tumor-suppressor genes. We developed the Plasmid-based microRNA Inhibitor System (PMIS), and it is the only miR inhibitor that can be used to generate transgenic mice to study the role of miRs during embryonic development and as disease models. The PMIS is very stable, efficient, has high specificity and affinity for specific miRs and is not toxic to cells or animals. We focused on miR-210 as it is increased in human colon cancer. The PMIS-miR-210 lentivirus construct and plasmid both express GFP. We made PMIS-miR-210 and PMIS-vector only stably transduced human SW620 colon cancer cell lines to determine the effect of inhibiting miR-210 on cell growth and phenotypes. The cells were transduced with the lentivirus and GFP expressing cells were FACS sorted and analyzed for growth. The PMIS-miR-210 cells proliferated very slowly (25% of the rate of PMIS-vector only cells). RNA-sequencing experiments and bioinformatics analyses revealed several new genes targeted by miR-210 and new pathways in colon tumor progression. We have identified a miR regulatory loop controlling the expression of other miRs. Nude mice were injected with PMIS-miR-210 or PMIS-vector stably expressing human colon cancer cells and 5 out of 8 mice with colon cancer cells expressing PMIS-miR-210 did not form tumors compared to the controls (PMIS-vector, 8 of 8 had tumors). To demonstrate the effectiveness of PMIS-miR-210 as a therapeutic, nude mice were injected with colon cancer cells and tumors were allowed to form (3 weeks), mice were then injected with naked plasmid DNA PMIS-miR-210 or vector control into and surrounding the solid tumor mass at several concentrations. No tumors were observed in 6 of the 10 mice injected with PMIS-miR-210 and the other 4 mice had small tumors compared to controls after 2 weeks of further growth (PMIS-vector, 10 of 10 had no effect on tumor growth). This is the first time that direct injection of a naked plasmid DNA expressing the PMIS microRNA inhibitor reduced or completely inhibited tumor growth, without using toxic delivery systems. The PMIS has great potential as a new therapeutic biological drug for cancer treatments. Support or Funding Information This work was supported by National Institutes of Health grant DE026433. A) Transduced colon cancer cells with either PMIS empty vector or PMIS-miR-210 were injected into nude mice and after 4 weeks the tumors were analyzed. B,C) Untreated colon cancer cells were put into nude mice and after 3 weeks, tumors were injected with PMIS empty vector or PMIS-miR-210, 2 weeks later the tumors were harvested and analyzed. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A method for using direct injection of plasmid DNA to study cis-regulatory element activity in F0Xenopus embryos and tadpoles

The ability to express exogenous reporter genes in intact, externally developing embryos, such as Xenopus, is a powerful tool for characterizing the activity of cis-regulatory gene elements during development. Although methods exist for generating transgenic Xenopus lines, more simplified methods for use with F0 animals would significantly speed the characterization of these elements. We discovered that injecting 2-cell stage embryos with a plasmid bearing a ϕC31 integrase-targeted attB element and two dual β-globin HS4 insulators flanking a reporter transgene in opposite orientations relative to each other yielded persistent expression with sufficiently high penetrance for characterizing the activity of the promoter without having to coinject integrase RNA. Expression began appropriately during development and persisted into swimming tadpole stages without perturbing the expression of the cognate endogenous gene. Coinjected plasmids having the same elements but expressing different reporter proteins were reliably coexpressed within the same cells, providing a useful control for variations in injections between animals. To overcome the high propensity of these plasmids to undergo recombination, we developed a method for generating them using conventional cloning methods and DH5α cells for propagation. We conclude that this method offers a convenient and reliable way to evaluate the activity of cis-regulatory gene elements in the intact F0 embryo.

Efficient and cost effective production of active-form human PKB using silkworm larvae.

Protein kinase B (PKB) also known as Akt is involved in many signal transduction pathways. As alterations of the PKB pathway are found in a number of human malignancies, PKB is considered an important drug target for cancer therapy. However, production of sufficient amounts of active PKB for biochemical and structural studies is very costly because of the necessity of using a higher organism expression system to obtain phosphorylated PKB. Here, we report efficient production of active PKBα using the BmNPV bacmid expression system with silkworm larvae. Following direct injection of bacmid DNA, recombinant PKBα protein was highly expressed in the fat bodies of larvae, and could be purified using a GST-tag and then cleaved. A final yield of approximately 1 mg PKBα/20 larvae was recorded. Kinase assays showed that the recombinant PKBα possessed high phosphorylation activity. We further confirmed phosphorylation on the activation loop by mass spectrometric analysis. Our results indicate that the silkworm expression system is of value for preparation of active-form PKBα with phosphorylation on the activation loop. This efficient production of the active protein will facilitate further biochemical and structural studies and stimulate subsequent drug development.

DNA/MVA Vaccines for HIV/AIDS.

Since the initial proof-of-concept studies examining the ability of antigen-encoded plasmid DNA to serve as an immunogen, DNA vaccines have evolved as a clinically safe and effective platform for priming HIV-specific cellular and humoral responses in heterologous “prime-boost” vaccination regimens. Direct injection of plasmid DNA into the muscle induces T- and B-cell responses against foreign antigens. However, the insufficient magnitude of this response has led to the development of approaches for enhancing the immunogenicity of DNA vaccines. The last two decades have seen significant progress in the DNA-based vaccine platform with optimized plasmid constructs, improved delivery methods, such as electroporation, the use of molecular adjuvants and novel strategies combining DNA with viral vectors and subunit proteins. These innovations are paving the way for the clinical application of DNA-based HIV vaccines. Here, we review preclinical studies on the DNA-prime/modified vaccinia Ankara (MVA)-boost vaccine modality for HIV. There is a great deal of interest in enhancing the immunogenicity of DNA by engineering DNA vaccines to co-express immune modulatory adjuvants. Some of these adjuvants have demonstrated encouraging results in preclinical and clinical studies, and these data will be examined, as well.

High Doses of Vascular Endothelial Growth Factor 165 Safely, but Transiently, Improve Myocardial Perfusion in No-Option Ischemic Disease

Gene therapy can induce angiogenesis in ischemic tissues. The aim of this study was to assess safety, feasibility, and results, both clinical and on myocardial perfusion, of gene therapy in refractory angina. This was a phase I/II, prospective, temporal-controlled series, clinical trial. Thirteen patients were maintained for minimum 6 months under optimized clinical management, and then received intramyocardial injections of 2000 μg plasmid vascular endothelial growth factor 165 and were followed by single-photon emission computed tomography (SPECT), treadmill tests, Minnesota quality of life questionnaire (QOL), and New York Heart Association (NYHA) functional plus Canadian Cardiovascular Society (CCS) angina classifications. There were no deaths, early or late. During the optimized clinical treatment, we observed worsening of rest ischemia scores on SPECT (p<0.05). After treatment, there was a transitory increase in myocardial perfusion at the third-month SPECT under stress (pre-operative [pre-op] 18.38 ± 7.51 vs. 3 months 15.31 ± 7.30; p<0.01) and at the sixth month under rest (pre-op 13.23 ± 7.98 vs. 6 months: 16.92 ± 7.27; p<0.01). One year after, there were improvements in treadmill test steps (pre-op 2.46 ± 2.07 vs.12 months 4.15 ± 2.23; p<0.01) and oxygen consumption (pre-op 7.66 ± 4.47 vs.12 months 10.89 ± 4.65; p<0.05), QOL (pre-op 48.23 ± 18.35 vs.12 months 28.31 ± 18.14; p<0.01) scores, and CCS (pre-op 3 [3-3.5] vs.12 months 2 [1-2.5]; p<0.01) and NYHA (pre-op 3 [3-3] vs. 2 [2-2] vs. 12 months 2 [1-2]; p<0.01) classes. Gene therapy demonstrated to be feasible and safe in this advanced ischemic cardiomyopathy patient sample. There were improvements in clinical evaluation parameters, and a transitory increase in myocardial perfusion detectable by SPECT scintigraphy. NCT00744315 http://clinicaltrials.gov/

Site‐targeted non‐viral gene delivery by direct DNA injection into the pancreatic parenchyma and subsequent in vivo electroporation in mice

The pancreas is considered an important gene therapy target because the organ is the site of several high burden diseases, including diabetes mellitus, cystic fibrosis, and pancreatic cancer. We aimed to develop an efficient in vivo gene delivery system using non-viral DNA. Direct intra-parenchymal injection of a solution containing circular plasmid pmaxGFP DNA was performed on adult anesthetized ICR female mice. The injection site was sandwiched with a pair of tweezer-type electrode disks, and electroporated using a square-pulse generator. Green fluorescent protein (GFP) expression within the injected pancreatic portion was observed one day after gene delivery. GFP expression reduced to baseline within a week of transfection. Application of voltages over 40 V resulted in tissue damage during electroporation. We demonstrate that electroporation is effective for safe and efficient transfection of pancreatic cells. This novel gene delivery method to the pancreatic parenchyma may find application in gene therapy strategies for pancreatic diseases and in investigation of specific gene function in situ.

Therapeutic potential of genes in cardiac repair

Cardiovascular diseases remain the primary reason of premature death and contribute to a major percentage of global patient morbidity. Recent knowledge in the molecular mechanisms of myocardial complications have identified novel therapeutic targets along with the availability of vectors that offer the chance for designing gene therapy technique for protection and revival of the diseased heart functions. Gene transfer procedure into the myocardium is demonstrated through direct injection of plasmid DNA or through the coronary vasculature using the direct or indirect delivery of viral vectors. Direct DNA injection to the myocardium is reported to be of immense value in research studies that aims at understanding the activities of various elements in myocardium. It is also deemed vital for investigating the effect of the myocardial pathophysiology on expression of the foreign genes that are transferred. Gene therapies have been reported to heal cardiac pathologies such as myocardial ischemia, heart failure and inherited myopathies in several animal models. The results obtained from these animal studies have also encouraged a flurry of early clinical trials. This translational research has been triggered by an enhanced understanding of the biological mechanisms involved in tissue repair after ischemic injury. While safety concerns take utmost priority in these trials, several combinational therapies, various routes and dose of delivery are being tested before concrete optimization and complete potential of gene therapy is convincingly understood.

Usage of Plasmid Vector Carrying vegf and fgf2 Genes after Spinal Cord Injury in Rats

Using rat model of spinal cord contusion injury at TVIII, we compared the effectiveness of immediate single transplantation of human mononuclear umbilical cord blood cells transfected with pBud-VEGF-FGF2 plasmid and immediate direct injection of the same plasmid into the lesion area. The results suggest that the delivery of therapeutic genes vegf and fgf2 in cells is more effective than direct injection of plasmid DNA with the same genes (judging from the number of myelinated fibers). Better tissue preservation and motor function recovery in experiments with direct injection of plasmid pBud-VEGF-FGF2 suggest that direct gene therapy seems to be an effective additional procedure to the method of gene delivery with transfected stem and progenitor cells.

Somatic gene transfer in European sea bass (Dicentrarchus labrax): Optimal fish conditions and exogenous control of foreign genes

The introduction of naked plasmid DNA in the somatic tissue of an adult organism was established as a system for transient gene transfer years ago. The genes contained in the plasmid are efficiently expressed and expression can be maintained over long periods of time. In the present work, we established this technique for an aquacultured marine fish, European sea bass (Dicentrarchus labrax). The target tissue was muscle and the delivery method used was direct injection of the plasmid DNA. The maximum expression of the introduced gene was detected 5days after the injection with an optimal dose of 25μg. The efficiency of this method had a clear dependence on water temperature and muscle condition. Different systems previously used in mammals for the improvement of DNA uptake in muscle cells have not worked properly in fish. However, we found that electroporation improved DNA uptake in fish reared at low temperatures. Use of this technique allowed us to evaluate the tetracycline-regulated gene expression system for its use in a marine fish for the first time. All together, these data provide the foundations for undertaking future applications for DNA vaccination or gene therapy in European sea bass.

In vivo gene transfer in mouse preimplantation embryos after intraoviductal injection of plasmid DNA and subsequent in vivo electroporation

The aim of this study was to investigate whether direct injection of nonviral DNA into the oviductal lumen and subsequent in vivo electroporation leads to in vivo gene transfer in mouse preimplantation embryos present within an oviduct, as an alternative to the pre-existing pronuclear microinjection-based transgenesis. With this technique, effects of expression of the gene of interest (GOI) on mouse preimplantation development can be monitored with relative ease. Superovulated 4-week-old B6C3F1 female mice (hybrids between C57BL/6N and C3H/HeN) were mated with adult B6C3F1 male mice. Two days later, females that had been identified as pregnant, based on the presence of copulation plugs, were injected with 1 µl of a solution containing an enhanced green fluorescent protein (EGFP) expression plasmid (0.5 µg) and 0.05% trypan blue. The entire oviduct was then electroporated using tweezer-type electrodes with 8 square-wave pulses of 50 V each with 50-ms duration. The next day, the 8-cell stage embryos were collected, and their number, morphology, and EGFP-derived fluorescence were recorded. Of the 12 oviducts (6 females used) examined, 3 contained fluorescent 8-cell stage embryos (33%, 19/58 tested), but the intensity of fluorescence varied among the embryos. In total, 10% (19/192 tested) of the embryos were fluorescent and the fluorescence was maintained in these embryos after 1 day of culture. However, the fluorescence disappeared in the late gestational stage fetuses, and the transgenes could not be detected. Our results indicate that it is possible to transfect in vivo preimplantation embryos, although the success rate appears to be relatively low and gene expression is transient. This technology may provide a new method for manipulating preimplantation embryos in vivo, by using, for example, Cre-mediated conditional DNA recombination.

Effect of direct injection of shark DNA into skeletal muscles on the productive performance characteristics of red tilapia (Oreochromis sp.) fed at different dietary regimes

The present work aims to study the effect of direct injection of shark ( Squalus acanthias L.) DNA into skeletal muscles of red tilapia ( Oreochromis sp.) fingerlings fed at different dietary regimes (three protein levels; 18, 22 and 26% each with two metabolizable energy levels; 244 and 260 kcal/100 g diet) on the productive performance. The results showed that growth performance, body composition and feed utilization of red tilapia injected with shark DNA had significant superiority (P ≤0.05) compared with non-injected fish. Red tilapia fed 22% protein diet had significant improving (P ≤0.05) most of the productive performance traits. Moreover, final body weight, weight gain, percent body weight increases, feed intake and protein retention percentage of red tilapia fed on diet containing 244 kcal/100 g, were significantly higher (P ≤0.05) than those of fish fed on diet containing 260 kcal/100 g. These data suggests that dietary protein can be spared down to 22% protein by direct injection of shark DNA into skeletal muscles of fish. Thus, feed costs can be reduced by a further reduction in dietary protein. Therefore, the result of the present work indicates a possible easy and rapid way for improving fish characteristics.

Extraordinary mullet growth through direct injection of foreign DNA

The present study aims to produce a genetically modified grey mullet, Mugil cephalus , with accelerated growth through direct injection of foreign DNA isolated from the liver of shark ( Squalus acanthias L.) or African catfish ( Clarias gariepinus ) into muscles of fingerlings fish at the dose of 40 μg/fish. The results show a significant (P≤0.05) improvement in most of the growth performance and body composition parameters of genetically modified grey mullet fingerlings injected with shark DNA compared to both genetically modified grey mullet injected with catfish DNA and the control fish, while the results of feed conversion ratio (FCR) and protein efficiency ratio (PER) indicate that fish injected with shark DNA or catfish DNA had significant (P≤0.05) superiority compared to their control. The results of the random amplified polymorphic DNA (RAPD) fingerprinting show highly genetic polymorphic percentage among grey mullet that received foreign DNA and their control using different random primers. This may be due to some fragments of foreign DNA randomly integrated into grey mullet genome. Therefore, the result indicates a possible easy and rapid way for improving fish characteristics. Keywords: Grey mullet, growth, foreign DNA, genetically modified

A strategy to obtain recombinant cell lines with high expression levels. Lentiviral vector-mediated transgenesis

BackgroundThe primary goal of any recombinant protein produc-tion is to achieve successful gene transfer and expres-sion in a target cell. There are two general categories ofdelivery vehicles/vectors employed in protein expressionprotocols. The first category includes the non-viral vec-tors, ranging from direct injection of DNA to complex-ing DNA with cationc lipds, polylysine, etc. The secondcategory comprises DNA and RNA viral vectors.Viruses have evolved specific mechanism to delivertheir genetic material to target cell nuclei. Virus mem-bers of family Retroviridae, e.g. retroviruses and lenti-viruses, are among the most widely used viral vectors.The use of lentiviral vectors has been increasing becausethe vector system has attractive features. Lentiviruseshave an advantage over retroviruses in that they caninfect both dividing and non-dividing cells and thereforehave attracted much attention regarding the potential asvectors for gene delivery/therapy. Once integrated intothe genome, recombinant cell lines are selected usingdifferent selection mechanisms.ResultsLentivirus particles were produced by simultaneous co-transfection of HEK 293T cells with four plasmids. Thepackaging construct (pMDLg/pRRE) [1], the VSV-G-expressing construct (pMD.G) [2], the Rev-expressingconstruct (pRSV-Rev) [1], and the self-inactivating (SIN)lentiviral vector construct containing the green fluores-cent protein (GFP) reporter gene (pLV-PLK-eGFP). Themedium containing lentiviral particles was collected 48h after transfection, clarified by centrifugation 10 min at2000 rpm and then stored at -80°C. To determine viraltiters, HEK 293T cells were seeded at 3 x 10

Osteoinduction by repeat plasmid injection of human bone morphogenetic protein‐2

Bone morphogenetic protein-2 (BMP-2) is an osteoinductive protein and is considered useful for the treatment of skeletal disorders. Previous studies using BMP-2 in clinical applications have encountered difficulties, including the lack of an efficient, safe, inexpensive and simple delivery system. The gene transfer approach is a promising option for utilizing BMP-2. Although viral vector-mediated gene transfer is efficient, safety concerns prevent its clinical application for common diseases. On the other hand, plasmid-based gene transfer is a safe method and can be harnessed for practical applications. A plasmid encoding human BMP-2 (pCAGGS-BMP-2) was used and injected repeatedly (one to eight times) into the skeletal muscle of mice at a divided dose. We compared the capability of osteoinduction in the skeletal muscle of mice after gene transfer by repeat injection. BMP-2 production was assessed via immunohistochemistry, and osteoinduction was evaluated using radiography, histology and biochemical assays. The BMP-2 gene was transferred into the skeletal muscle of mice by repeat injection using pCAGGS-BMP-2. Mature bone was frequently observed in mice injected repeatedly with pCAGGS-BMP-2 at a divided dose. This confirms that, if the total dose is fixed, repeat injection with pCAGGS-BMP-2 at a divided dose causes osteoinduction more frequently in the skeletal muscle of mice. These results suggest the possibility of the effective clinical use of human BMP-2 gene therapy by direct DNA injection, and facilitate the clinical application of BMP-2 gene therapy.

Defining environmental risk assessment criteria for genetically modified fishes to be placed on the EU market

Defining environmental risk assessment criteria for genetically modified fishes to be placed on the EU market

Injection site‐dependent induction of immune response by DNA vaccine: comparison of skin and spleen as a target for vaccination

The antigen-specific immune response is dependent not only on the properties of the antigens, but also on their encounter with antigen-presenting cells. A previous study showed that the spleen produced a large amount of transgenes after direct tissue injection of plasmid DNA. In addition, the spleen is the largest organ in the lymphatic system and contains a variety of types of immune cells, including lymphocytes, macrophages and dendritic cells. Thus, it can be a promising target for DNA vaccination. Tissue-dependent properties of transgene expression were examined using a plasmid vector expressing firefly luciferase. Mice received injections of pCMV-Luc into the dorsal skin or spleen followed by electroporation, and the luciferase activity was measured 6 h after injection. Then, plasmids expressing a model antigen ovalbumin (pCMV-OVA) or its typical major histocompatibility complex class I-restricted epitope SIINFEKL (pPep-ER) were injected into C57BL/6 mice twice at an interval of 1 week. Seven days after the second immunization, OVA-specific humoral and cellular immune responses were evaluated. The spleen produced a larger amount of transgenes than the skin after direct tissue injection of plasmid DNA. However, intradermal injection of plasmid DNA resulted in a larger amount of OVA-specific antibodies and a greater cytotoxic T lymphocyte response compared to intrasplenic injection. In addition, intradermal immunization with either pCMV-OVA or pPep-ER generated more protective effects against EG7-OVA tumor challenge. The results obtained in the present study indicate that the spleen is unlikely to be a good target for immunization despite the presence of a large number of lymphocytes and efficient production of transgenes.

Suppression of teratocarcinoma growth by soluble TRAIL gene driven by the progression-elevated gene-3 promoter

A plasmid expressing the soluble tumor necrosis factor (TNF)-related apoptosis-inducing ligand sTRAIL amino acids 114-281 of TRAIL), driven by rat progression-elevated gene-3 (rPEG) promoter was constructed and evaluated. Transfection of embryonal carcinoma (EC) cells with the plasmid resulted in significant cellular apoptosis and elevated expression of death receptor 4 (DR4) and death receptor 5 (DR5). Direct intratumoral injection of DNA:liposome complexes suppressed tumor growth significantly and prolonged the survival of teratocarcinoma-bearing mice. Histological examination and serum analyses showed the absence of detectable toxicity in all examined tissues including liver. Our results demonstrate that sTRAIL gene expression driven by the rPEG promoter may enable effective gene therapy against teratocarcinoma.