Levels Of Foreign Gene Expression Research Articles

Engineering the Staple Oil Crop Brassica napus Enriched with α-Linolenic Acid Using the Perilla FAD2-FAD3 Fusion Gene.

Modern people generally suffer from α-linolenic acid (ALA) deficiency, since most staple food oils are low in ALA content. Thus, the enhancement of ALA in staple oil crops is of importance. In this study, the FAD2 and FAD3 coding regions from the ALA-king species Perilla frutescens were fused using a newly designed double linker LP4-2A, driven by a seed-specific promoter PNAP, and engineered into a rapeseed elite cultivar ZS10 with canola quality background. The mean ALA content in the seed oil of PNAP:PfFAD2-PfFAD3 (N23) T5 lines was 3.34-fold that of the control (32.08 vs 9.59%), with the best line being up to 37.47%. There are no significant side effects of the engineered constructs on the background traits including oil content. In fatty acid biosynthesis pathways, the expression levels of structural genes as well as regulatory genes were significantly upregulated in N23 lines. On the other hand, the expression levels of genes encoding the positive regulators of flavonoid-proanthocyanidin biosynthesis but negative regulators of oil accumulation were significantly downregulated. Surprisingly, the ALA level in PfFAD2-PfFAD3 transgenic rapeseed lines driven by the constitutive promoter PD35S was not increased or even showed a slight decrease due to the lower level of foreign gene expression and downregulation of the endogenous orthologous genes BnFAD2 and BnFAD3.

Agrobacterium-Mediated Genetic Transformation of Larix kaempferi (Lamb.) Carr. Embryogenic Cell Suspension Cultures and Expression Analysis of Exogenous Genes

A simple and efficient protocol for the genetic transformation of Japanese larch (Larix kaempferi (Lamb.) Carr.) was developed by altering the infection method and duration and the bacterial removal process. More than 600 hygromycin-resistant embryonal masses with the vector pCAMBIA1301 were obtained, with an average of 20 transgenic lines per gram of fresh weight. Nine hygromycin-resistant transformation events (designated P1–P9) were analyzed using PCR, quantitative real-time PCR, and histochemical β-glucuronidase (GUS) assays. The GUS transcript abundance in each transformed cell line ranged from 101 to 103 magnitudes, with a maximum abundance of 2.89 × 103. In addition, the pLaTCTP::GUS vector, which contains GUS under the control of the L. kaempferi LaTCTP promoter, led to constitutive expression of GUS in embryonal-suspensor mass and somatic embryos. The transcript abundance of the exogenous genes HPT and GUS, driven by the CaMV 35S or LaTCTP promoter, ranged from 101 to 104, which was equivalent to genes with moderate and low abundances in Japanese larch. The relatively low expression levels of exogenous genes in transformants might reflect the large genome of Japanese larch. Additional transgenic cell lines need to be screened to obtain transformants with higher expression levels of foreign genes for further functional research in Japanese larch.

Expression of Two Foreign Genes by a Newcastle Disease Virus Vector From the Optimal Insertion Sites through a Combination of the ITU and IRES-Dependent Expression Approaches.

Many Newcastle disease virus (NDV) strains have been developed as vectors to express a foreign gene (FG) for vaccine and cancer therapy purposes. The non-coding region between the phosphoprotein (P) and matrix protein (M) genes and the non-coding region behind the NP gene open reading frame (ORF) in the NDV genome have been identified as the optimal insertion sites for efficient FG expression through the independent transcription unit (ITU) and the internal ribosomal entry site (IRES) dependent expression approaches, respectively. To date, however, the majority of these NDV vectors express only a single or two FGs from suboptimal insertion sites in the NDV genome, obtaining various levels of FG expression. To improve the FG expression, we generated NDV LaSota vaccine strain-based recombinant viruses expressing two FGs, GFP, and RFP, from the identified optimal insertion sites through a combination of the ITU and IRES-dependent approaches. Biological assessments of the recombinant viruses indicated that the recombinants expressing two FGs were slightly attenuated with approximately one order of magnitude lower in virus titers when compared to the viruses containing a single FG. The FG expression efficiencies from the two-FG viruses were also lower than those from the single-FG viruses. However, the expression of two FGs from the optimal insertion sites was significantly (p < 0.05) higher than those from the suboptimal insertion sites. The expressions of FGs as monocistronic ITU were approximately 4-fold more efficient than those expressed by the bicistronic IRES-dependent approach. These results suggest that the NDV LaSota vector could efficiently express two FGs from the identified optimal insertions sites. The ITU strategy could be used for “vectoring” FGs in circumstances where high expression of gene products (e.g., antigens) is warranted, whereas, the IRES-dependent tactic might be useful when lower amounts of IRES-directed FG products are needed.

Elevated methionine content in soybean seed by overexpressing maize β-zein protein

Soybean provides superior and readily available protein for human and livestock. However, nutritional value of soybean is limited due to the deficiency of an essential amino acid, methionine. To improve total methionine content of soybean, a methionine-rich seed storage protein, β-zein, was introduced into soybean cultivar ’Jack’ under the control of legumin B4 promoter or CaMV 35S promoter. Totally 4 T3 transgenic lines exhibited higher expression levels of foreign genes, and legumin B4 promoter directed a stronger accumulation of β-zein protein than CaMV 35S promoter. Compared to wild type plant, total methionine content in transgenic soybean seeds significantly increased by up to approximately 15%. Although the introduction of β-zein gene improved total methionine content, the level was negligible compared to native soybean storage proteins, implying that the inadequate soluble methionine is the limiting factor. Based on these observations, a new strategy for simultaneously increasing the “source” and “sink” of methionine metabolism is proposed to further improvement of total methionine content in soybean seed.

Temporal characterization of protein production levels from baculovirus vectors coding for GFP and RFP genes under non-conventional promoter control.

The ease of use and versatility of the Baculovirus Expression Vector System (BEVS) has made it one of the most widely used systems for recombinant protein production However, co-expression systems currently in use mainly make use of the very strong very late p10 and polyhedron (polh) promoters to drive expression of foreign genes, which does not provide much scope for tailoring expression ratios within the cell. This work demonstrates the use of different Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) promoters to control the timing and expression of two easily traceable fluorescent proteins, the enhanced green fluorescent protein (eGFP), and a red fluorescent protein (DsRed2) in a BEVS co-expression system. Our results show that gene expression levels can easily be controlled using this strategy, and also that modulating the expression level of one protein can influence the level of expression of the other protein within the system, thus confirming the concept of genes "competing" for limited cellular resources. Plots of "expression ratios" of the two model genes over time were obtained, and may be used in future work to tightly control timing and levels of foreign gene expression in an insect cell co-expression system.

PhiC31 integrase induces efficient site-specific recombination in the Capra hircus genome.

Streptomyces phage φC31 integrase induces efficient site-specific recombination capable of integrating exogenous genes at pseudo attP sites in human, mouse, rat, rabbit, sheep, Drosophila, and bovine genomes. However, the φC31-mediated recombination between attB and the corresponding pseudo attP sites has not been investigated in Capra hircus. Here, we identified eight pseudo attP sites located in the intron or intergenic regions of the C. hircus genome, and demonstrated different levels of foreign gene expression after φC31 integrase-mediated integration. These pseudo attP sites share similar sequences with each other and with pseudo attP sites in other mammalian genomes, and these are associated with a neighboring consensus motif found in other genomes. The application of the φC31 integrase system in C. hircus provides a new option for genetic engineering of this economically important goat species.

Innate Immune Response Induced by Baculovirus Attenuates Transgene Expression in Mammalian Cells

The baculovirus Autographa californica nucleopolyhedrovirus (AcNPV) has been widely used to achieve a high level of foreign gene expression in insect cells, as well as for efficient gene transduction into mammalian cells without any replication. In addition to permitting efficient gene delivery, baculovirus has been shown to induce host innate immune responses in various mammalian cells and in mice. In this study, we examined the effects of the innate immune responses on gene expression by recombinant baculoviruses in cultured cells. The reporter gene expression in IRF3-deficient mouse embryonic fibroblasts (MEFs) infected with the recombinant baculovirus was shown to be enhanced in accordance with the suppression of beta interferon (IFN-β) production. Furthermore, efficient gene transduction by the recombinant baculovirus was achieved in MEFs deficient for stimulator of interferon genes (STING), TANK binding kinase 1 (TBK1), IFN regulatory factor 3 (IRF3), or IFN-β promoter stimulator 1 (IPS-1), but not in those deficient for IRF7, MyD88, or Z-DNA binding protein 1 (ZBP1)/DAI. Enhancement of gene expression by the recombinant baculovirus was also observed in human hepatoma cell lines replicating hepatitis C virus (HCV), in which innate immunity was impaired by the cleavage of IPS-1 by the viral protease. In addition, infection with the recombinant baculovirus expressing the BH3-only protein, BIMS, a potent inducer of apoptosis, resulted in a selective cell death in the HCV replicon cells. These results indicate that innate immune responses induced by infection with baculovirus attenuate transgene expression, and this characteristic might be useful for a selective gene transduction into cells with impaired innate immunity arising from infection with various viruses.

A New Method to Determine Antigen-Specific CD8+ T Cell Activity in Vivo by Hydrodynamic Injection.

Hydrodynamic tail vein (HTV) delivery is a simple and rapid tail vein injection method of a high volume of naked plasmid DNA resulting in high levels of foreign gene expression in organs, especially the liver. Compared to other organs, HTV delivery results in more than a 1000-fold higher transgene expression in liver. After being bitten by malaria-infected mosquitoes, malaria parasites transiently infect the host liver and form the liver stages. The liver stages are known to be the key target for CD8+ T cells that mediate protective anti-malaria immunity in an animal model. Therefore, in this study, we utilized the HTV delivery technique as a tool to determine the in vivo cytotoxic effect of malaria antigen-specific CD8+ T cells. Two weeks after mice were immunized with recombinant adenoviruses expressing malarial antigens, the immunized mice as well as naïve mice were challenged by HTV delivery of naked plasmid DNA co-encoding respective antigen together with luciferase using dual promoters. Three days after the HTV challenge, non-invasive whole-body bioluminescent imaging was performed. The images demonstrate in vivo activity of CD8+ T cells against malaria antigen-expressing cells in liver.

ウイルス感染による宿主自然免疫応答の解析と感染制御への応用

The baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) has been widely used not only to achieve a high level of foreign gene expression in insect cells but also for efficient gene transduction into mammalian cells without any replication. In addition to the efficient gene delivery, baculovirus has been shown to induce host innate immune responses in various mammalian cells and in mice. The baculovirus has abundant CpG motifs in the viral genome and is capable of inducing pro-inflammatory cytokines and interferons (IFNs) through Toll-like receptor (TLR)-dependent and -independent signaling pathways in a cell-type-specific manner. The cytoplasmic helicase proteins RIG-I (retinoic-acid-inducible protein I) and MDA5 (melanoma-differentiation-associated gene 5) have been identified as viral dsRNA detectors and the adaptor IPS-1 (IFN-beta promoter stimulator-1) interacts with RIG-I and MDA5 to facilitate type-I IFN production mediated interferon regulatory factor 3 (IRF3) and 7 (IRF7). These helicases and IPS-1, however, were not essential for the type-I IFN and inflammatory cytokine responses to baculovirus. The baculovirus also has a strong adjuvant activity, and recombinant baculoviruses encoding neutralization epitopes elicit protective immunity in mice. This review deals with the current status of our knowledge of the induction of host innate immune responses by baculovirus and discusses the future prospects for baculovirus vectors.

Lentiviral Based Gene Transduction and Promoter Studies in Human Hematopoietic Stem Cells (hHSCs)

Human hematopoietic stem cells (hHSCs) have enormous potential for clinical use in cell-based therapies, especially as a gene delivery system. Moreover, lentiviral transduction in stem cells is very often associated with low transduction efficiency and low levels of foreign gene expression. Therefore, it is important to analyze vector and promoter systems that can generate robust foreign gene expression in these cells. In this study, we evaluated and compared the ability of different commercially available promoters to drive the expression of exogenous reporter genes in hHSCs and evaluated the effect of different doses of stem cell growth factors on the expression of transgenes. We used lentivirus based vector system carrying the following promoters: 1) Human cytomegalovirus (CMV) promoter, 2) Simian virus 40 (SV40) promoter, 3) mammalian Ubiquitin C (UBC) promoter and 4) cellular polypeptide chain elongation factor 1 alpha (EF1) promoter. EF1 and CMV promoters robustly drove the expression of green fluorescence protein (GFP) reporter gene, while SV40 and UBC promoters induced very low level of GFP expression. Lentivectors containing EF1 and CMV promoters showed high-level stable GFP expression in human cord blood stem cells for 6 weeks period after post transduction. CD133+ hHSCs stimulated with higher concentration of growth factors exhibited enhancement of transduction rate. Cord blood derived CD133+ hHSCs could be effectively transduced with lentivectors under CMV or EF-1 promoters for the expression of foreign gene.

Improved foreign gene expression in plants using a virus‐encoded suppressor of RNA silencing modified to be developmentally harmless

Endeavours to obtain elevated and prolonged levels of foreign gene expression in plants are often hampered by the onset of RNA silencing that negatively affects target gene expression. Plant virus-encoded suppressors of RNA silencing are useful tools for counteracting silencing but their wide applicability in transgenic plants is limited because their expression often causes harmful developmental effects. We hypothesized that a previously characterized tombusvirus P19 mutant (P19/R43W), typified by reduced symptomatic effects while maintaining the ability to sequester short-interfering RNAs, could be used to suppress virus-induced RNA silencing without the concomitant developmental effects. To investigate this, transient expression in Nicotiana benthamiana was used to evaluate the ability of P19/R43W to enhance heterologous gene expression. Although less potent than wt-P19, P19/R43W was an effective suppressor when used to enhance protein expression from either a traditional T-DNA expression cassette or using the CPMV-HT expression system. Stable transformation of N.benthamiana yielded plants that expressed detectable levels of P19/R43W that was functional as a suppressor. Transgenic co-expression of green fluorescent protein (GFP) and P19/R43W also showed elevated accumulation of GFP compared with the levels found in the absence of a suppressor. In all cases, transgenic expression of P19/R43W caused no or minimal morphological defects and plants produced normal-looking flowers and fertile seed. We conclude that the expression of P19/R43W is developmentally harmless to plants while providing a suitable platform for transient or transgenic overexpression of value-added genes in plants with reduced hindrance by RNA silencing.

Host Innate Immune Responses Induced by Baculovirus in Mammals

The baculovirus Autographa californica nuclear polyhedrosis virus has been widely used not only to achieve a high level of foreign gene expression in insect cells but also for efficient gene transduction into mammalian cells without any replication. In addition to the efficient gene delivery, baculovirus has been shown to induce host innate immune responses in various mammalian cells and in mice. The baculovirus has abundant CpG motifs in the viral genome and is capable of inducing pro-inflammatory cytokines and interferons through Toll-like receptor-dependent and -independent signaling pathways in a cell-type-specific manner. The baculovirus also has a strong adjuvant activity, and recombinant baculoviruses encoding neutralization epitopes elicit protective immunity in mice. This review deals with the current status of our knowledge of the induction of host innate immune responses by baculovirus and discusses the future prospects for baculovirus vectors.

PiggyBac Transposon-mediated Long-term Gene Expression in Mice

Transposons are promising systems for somatic gene integration because they can not only integrate exogenous genes efficiently, but also be delivered to a variety of organs using a range of transfection methods. piggyBac (PB) transposon has a high transposability in mammalian cells in vitro, and has been used for genetic and preclinical studies. However, the transposability of PB in mammalian somatic cells in vivo has not been demonstrated yet. Here, we demonstrated PB-mediated sustained gene expression in adult mice. We constructed PB-based plasmid DNA (pDNA) containing reporter [firefly and Gaussia luciferase (Gluc)] genes. Mice were transfected by injection of these pDNAs using a hydrodynamics-based procedure, and the conditions for high-level sustained gene expression were examined. Consequently, gene expressions were sustained over 2 months. Our results suggest that PB is useful for organ-selective somatic integration and sustained gene expression in mammals, and will contribute to basic genetic studies and gene therapies.

Systemic Correction of Storage Disease in MPS I NOD/SCID Mice Using the Sleeping Beauty Transposon System

The Sleeping Beauty (SB) transposon system is a nonviral vector that directs transgene integration into vertebrate genomes. We hydrodynamically delivered SB transposon plasmids encoding human alpha-L-iduronidase (hIDUA) at two DNA doses, with and without an SB transposase gene, to NOD.129(B6)-Prkdc(scid) IDUA(tm1Clk)/J mice. In transposon-treated, nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice with mucopolysaccharidosis type I (MPS I), plasma IDUA persisted for 18 weeks at levels up to several hundred-fold wild-type (WT) activity, depending on DNA dose and gender. IDUA activity was present in all examined somatic organs, as well as in the brain, and correlated with both glycosaminoglycan (GAG) reduction in these organs and level of expression in the liver, the target of transposon delivery. IDUA activity was higher in the treated males than in females. In females, omission of transposase source resulted in significantly lower IDUA levels and incomplete GAG reduction in some organs, confirming the positive effect of transposition on long-term IDUA expression and correction of the disease. The SB transposon system proved efficacious in correcting several clinical manifestations of MPS I in mice, including thickening of the zygomatic arch, hepatomegaly, and accumulation of foamy macrophages in bone marrow and synovium, implying potential effectiveness of this approach in treatment of human MPS I.

The Distribution of mRNA Expression and Protein after Hydrodynamic Injection of Transgene in Mice

The hydrodynamic method by rapid intravenous injection of a large volume of plasmid DNA is known to be an efficient and liver-specific method of in vivo gene delivery and achieves high levels of foreign gene expression, particularly in hepatocytes. Low transgene activities have also been observed in other organs such as the spleen and lung; however, the expression profiles of mRNA and protein are still unknown. Therefore, we investigated the localization of luciferase mRNA by in situ hybridization and luciferase activity in mice after transfection of pCMV-luc encoding the luciferase gene under the control of cytomegalo virus (CMV) promoter. We found that hydrodynamic injection effectively induced mRNA expression of the transgene only in the liver although transgene activities were observed in other organs. The transgene activity observed in other organs may be due to leakage from hepatocyte gene expression by transient increase in the permeability of the hepatocyte cellular membrane caused by increased pressure by hydrodynamic injection.

Electroporation-mediated transfer of plasmid DNA encoding IL-10 attenuates orthotopic tracheal allograft stenosis in rats

Background Electroporation has been shown to increase the efficacy of intramuscular injection of plasmid DNA, resulting in a higher level of foreign gene expression. Using this technique, we examined the effect of viral IL-10 gene transfer on the prevention of tracheal allograft stenosis in an animal model. Methods On the day of tracheal transplantation, recipient Lewis rats were intramuscularly injected with either plasmid pCAGGS-LacZ or plasmid pCAGGS-viral IL-10, followed immediately by electroporation. Tracheas from Brown Norway donors were transplanted into the backs of Lewis recipients, and the histology of the grafts were assessed 2 and 4 weeks after transplantation. Results The serum level of IL-10 peaked at 2000 pg/ml one day after injection; the level then slowly decreased, but was maintained above 1000 pg/ml until 8 days after injection. At Day 28, the airway lumina of the tracheal allografts were almost completely obliterated by fibroproliferative tissue in the control pCAGGS-LacZ-treated rats. In rats injected once with pCAGGS-viral IL-10, luminal obliteration was significantly decreased compared with the control pCAGGS-LacZ-treated rats (mean luminal opening 46.8% vs 0% p < 0.05). The loss of epithelial cells lining the airway was also decreased in the IL-10-treated group (mean epithelial coverage 42% vs 5% p < 0.05). Multiple injections with pCAGGS-viral IL-10 did not further improve the histological changes. Conclusion IL-10 gene transfer by intramuscular injection using electroporation attenuated tracheal allograft stenosis associated with mild epithelial injury.

Hydrodynamics‐based delivery of plasmid DNA encoding CTLA4‐Ig prolonged cardiac allograft survival in rats

Although gene therapy using plasmid vectors is thought to be safer compared with viral vectors, poor efficacy of gene transfer is the obstacle preventing wide application of plasmid vectors. However, high levels of foreign gene expression have been achieved by rapid tail vein injection of a large volume of a plasmid DNA solution into rats. Using this technique, we examined the effect of rat CTLA4-Ig gene transfer on prevention of cardiac allograft rejection in this animal model. Recipient Lewis rats were injected with either plasmid pCAGGS-CTLA4-Ig-Glu-tag as a treatment vector or plasmid pCAGGS-signal peptide (SP)-Ig as a control vector by hydrodynamics-based delivery technique on the day before heart transplantation. Hearts from Brown Norway donors were transplanted into the neck of Lewis recipients and graft survival was assessed. The plasma level of CTLA4-Ig reached a peak of nearly 5 microg/mL 1 day after injection, and then slowly decreased but still remained above 0.9 microg/mL until 100 days after injection. The recipient rats treated with the control vector and untreated rats rejected cardiac allografts within 7 days. On the other hand, the median survival time of the grafts treated with pCAGGS-CTLA4Ig-Glu-tag was more than 100 days. Histological examination revealed that long-term survival allografts contained fewer infiltrating lymphocytes. The serum from recipients with long-term survival allograft suppressed allogenic mixed lymphocyte reaction. CTLA4-Ig gene transfer by means of tail vein injection of plasmid DNA into a recipient rat resulted in remarkable prolongation of cardiac allograft survival with persistent plasma level of CTLA4-Ig protein.

Baculovirus Vector for Gene Delivery and Vaccine Development

The baculovirus Autographa californica multiple nucleopolyhedrovirus has been widely used not only to acheive a high level of foreign gene expression in insect cells, but also for efficient gene transduction into mammalian cells. Recombinant and pseudotyped baculoviruses possessing chimeric or foreign ligands have been constructed to improve the efficiency of gene transduction and to confer specificity for gene delivery into mammalian cells, respectively. Baculoviral DNA CpG motifs induce proinflammatory cytokines through a Toll-like receptor (TLR9)/MyD88-dependent signaling pathway. Other baculovirus components produce type I interferons via a TLR-independent pathway. Baculovirus exhibits a strong adjuvant property and recombinant baculoviruses encoding microbial antigens elicit antibodies to the antigens and provide protective immunity in mice. This review deals with recent progress in the application of baculovirus vectors to gene delivery and vaccine development, and discusses the future prospects of baculovirus vectors.

Pseudo-hydrodynamic Delivery of Helper-dependent Adenoviral Vectors into Non-human Primates for Liver-directed Gene Therapy

Helper-dependent adenoviral vectors (HDAds) are attractive for liver-directed gene therapy because they can mediate long-term, high-level transgene expression without chronic toxicity. However, systemic delivery requires high vector doses for efficient hepatic transduction, resulting in dose-dependent acute toxicity. Clearly, strategies to improve hepatic transduction with low vector doses are needed. In this regard, we have previously shown that hydrodynamic injection of helper-dependent adenoviral vectors into mice results in increased hepatic transduction, reduced systemic vector dissemination, and reduced pro-inflammatory cytokines compared with conventional injection and thus has the potential to improve dramatically the therapeutic index of helper-dependent adenoviral vectors. Unfortunately, the rapid, large-volume injection used in this method cannot be applied to larger animals. Therefore, we have developed a novel balloon occlusion catheter-based method to mimic hydrodynamic injection of helper-dependent adenoviral vectors into non-human primates that does not require rapid, large-volume injection. Using a low, clinically relevant vector dose, this minimally invasive method results in high-efficiency hepatic transduction with minimal toxicity and stable long-term transgene expression for at least 413 days.

Hydrodynamic Limb Vein Delivery of a Xenogeneic DNA Cancer Vaccine Effectively Induces Antitumor Immunity

Tumor-associated antigens (TAA) are typically poorly immunogenic "self" antigens. An effective strategy to break tolerance and induce antitumor immunity is by genetic vaccination, employing the orthologous TAA-sequence from a different species. We recently developed a clinically relevant approach for intravascular hydrodynamic limb vein (HLV) delivery of nucleic acids to skeletal muscle. Using the human gp100 xenogeneic TAA in the murine B16 melanoma model, we show that genetic vaccination of mice by HLV plasmid DNA delivery was highly effective at breaking tolerance against the homologous murine gp100 (mgp100) TAA and induced prophylactic antitumor protection. HLV vaccination resulted in an anti-hgp100 humoral and cellular response, with 4-5% of CD8(+) T cells being gp100(25-33)-epitope-specific. Vaccinated animals demonstrated in vivo cytolytic activity against human and mgp100(25-33) peptide-pulsed targets. Antitumor immunity could be adoptively transferred by splenocytes from human gp100-vaccinated animals. Furthermore, a durable antitumor memory response was established as approximately 3% of CD8(+) T cells were gp100(25-33) antigen-specific in mice 6 months after vaccination. Following a single HLV human gp100 DNA boost, this level increased to approximately 17% and protected animals from subsequent B16 tumor rechallenge. Our results warrant further consideration of HLV as a clinically relevant method for cancer gene therapy.