Regulation Of Transgene Expression Research Articles

Temporal and spatial control of transgene expression using laser induction of the hsp70promoter

BackgroundPrecise temporal and spatial regulation of transgene expression is a critical tool to investigate gene function in developing organisms. The most commonly used technique to achieve tight control of transgene expression, however, requires the use of specific DNA enhancers that are difficult to characterize in non-model organisms. Here, we sought to eliminate the need for this type of sequence-based gene regulation and to open the field of functional genetics to a broader range of organisms.ResultsWe have developed a new laser mediated method to heat shock groups of cells that provides precise spatio-temporal control of gene expression without requiring knowledge of specific enhancer sequences. We tested our laser-system in a transgenic line of Bicyclus anynana butterflies containing the EGFP reporter gene attached to the heat sensitive hsp70 promoter of Drosophila melanogaster. Whole organismal heat shocks demonstrated that this Drosophila promoter can drive gene expression in butterflies, and the subsequent laser heat shocks showed that it was possible to activate cell-specific gene expression in very precise patterns on developing pupal wings.ConclusionThis laser-mediated gene expression system will enable functional genetic investigations, i.e., the ectopic expression of genes and their knock-down in targeted groups of cells in model and non-model organisms with little or no available regulatory data, as long as a compatible heat-shock promoter is used and the target tissue is accessible to a laser beam. This technique will also be useful in evolutionary developmental biology as it will enable the study of the evolution of gene function across a variety of organisms.

Immune Regulation of Transgene Expression in the Brain: B Cells Regulate an Early Phase of Elimination of Transgene Expression from Adenoviral Vectors

Cellular immune mechanisms that regulate viral gene expression within infected brain cells remain poorly understood. Previous work has shown that systemic immunization against adenovirus after vector delivery to the brain results in complete loss of brain cells infected by adenoviral vectors. Although T cells play an important role in this process, we demonstrate herein that B cells also significantly regulate transgene expression from the CNS. After the systemic immunization against adenovirus of animals injected via the brain with an adenoviral vector 30 days earlier, we uncovered substantial infiltration by CD19+ B cells of the area of the brain transduced by the virus. This suggests the involvement of B cells in the adaptive immune response-mediated loss of transduced cells from the brain. Confocal analysis of these brains demonstrated physical contacts between transduced brain cells and CD19+ cells. To test the hypothesis that B cells play a causal role in the loss of infected cells from the brain, we demonstrated that animals devoid of B cells were unable to eliminate transgene expression at early time points after immunization. This demonstrates that B cells play a necessary role in the loss of transgene expression at early, but not late, time points postimmunization. Thus, these data have important implications for our understanding of the role of B cells as immune effectors during the immune-mediated clearance of viral infections from the CNS, and also for understanding mechanisms operating in brain autoimmunity, as well as for the potential safety of clinical gene therapy for brain diseases.

4:22 97. Regulation of Transgene Expression Using an Inducible System for Improved Safety of Intradiscal Gene Therapy

4:22 97. Regulation of Transgene Expression Using an Inducible System for Improved Safety of Intradiscal Gene Therapy

Tetracycline‐inducible gene expression in nuclear transfer embryos derived from porcine fetal fibroblasts transformed with retrovirus vectors

A critical problem of transgenic livestock production is uncontrollable constitutive expression of the foreign gene, which usually results in serious physiological disturbances in transgenic animals. One of the best solutions for this problem may be use of controllable gene expression system. In this study, using retrovirus vectors designed to express the enhanced green fluorescent protein (EGFP) gene under the control of the tetracycline-inducible promoter, we examined whether the expression of the transgene could be controllable in fibroblast cells and nuclear transfer (NT) embryos of porcine. Transformed fibroblast cells were cultured in medium supplemented with or without doxycycline (a tetracycline analog) for 48 hr, and the induction efficiency was measured by comparing EGFP gene expression using epifluorescence microscopy and Western and Northern blot analyses. After the addition of doxycycline, EGFP expression increased up to 17-fold. The nuclei of transformed fibroblast cells were transferred into enucleated oocytes. Fluorescence emission data revealed strong EGFP gene expression in embryos cultured with doxycycline, but little or no expression in the absence of the antibiotic. Our results demonstrate the successful regulation of transgene expression in porcine nuclear transfer embryos, and support the application of an inducible expression system in transgenic pig production to solve the inherent problems of side-effects due to constitutive expression of the transgene.

Applications of Gene Therapy and Adult Stem Cells in Bone Bioengineering

Bone tissue engineering is an emerging field, that could become a main therapeutic strategy in orthopedics in coming years. While bone has regenerative abilities that enable the self repair and regeneration of fractures, there are extreme situations in which the extent of bone loss is too large for complete regeneration to occur. In order to achieve bone regeneration, osteogenic genes (mainly from the bone morphogenetic protein family) can be delivered either directly into the target tissue, or by using adult stem cells, which are later implanted into the target site. Engineered adult stem cells combined with biodegradable polymeric scaffolds can be implanted into target sites, with or without ex vivo culture period. Several important factors influence the success of bone engineering approaches including: choice of cell and scaffold, the vector used in order to deliver the osteogenic gene, and the osteogenic gene itself. Cutting-edge imaging technologies, bioinformatics-based analysis of gene expression and exogenous regulation of transgene expression are among the tools that are being used to optimize and control bone formation in vivo. In this review we have attempted to provide an overview of the main factors that should be considered when utilizing adult stem cells and gene therapy strategies to regenerate bone defects or to promote new bone formation in vivo.

Inflammatory cytokine regulation of transgene expression in human fibroblast‐like synoviocytes infected with adeno‐associated virus

An ideal gene transfer vector for chronic inflammatory diseases such as rheumatoid arthritis (RA) would provide local transgene expression only when the disease is active. To determine whether adeno-associated virus (AAV) possesses this ability, the effects of inflammatory cytokines on transgene expression were evaluated in human RA fibroblast-like synoviocytes (FLS). Human FLS were infected with AAV in the presence or absence of inflammatory cytokines or synovial fluid obtained from patients with RA. Transgene expression was monitored by either enzyme-linked immunosorbent assay or flow cytometry. Transgene messenger RNA (mRNA) was measured by real-time quantitative reverse transcription-polymerase chain reaction. Inflammatory cytokines increased transgene expression in FLS by up to 60-fold. Synovial fluid from patients with RA, but not from patients without arthritis, was also able to increase expression in synoviocytes. Protein expression correlated with transgene mRNA levels. The enhanced expression required the continued presence of cytokines because, upon removal, transgene expression returned to baseline levels. Expression could be repeatedly reinduced by reexposure to cytokines. The effect was not promoter specific and was demonstrated to be phosphatidylinositol 3-kinase-dependent. These results suggest that expression of a therapeutic transgene can be controlled by the presence of inflammation following AAV gene transfer, making it an attractive vector for chronic inflammatory diseases such as RA.

Delivery of Large Genomic DNA Inserts > 100 kb Using HSV-1 Amplicons

The principal aim of gene therapy for recessive genetic diseases is to supplement the loss of function of an endogenous gene. For the treatment of many diseases regulation of transgene expression at physiological levels, expression of multiple splice variants, and correct tissue specificity are of utmost importance for effective therapy. We therefore believe the use of a complete genomic locus, in which the native promoter and regulatory regions drive and control expression, is an elegant and effective alternative to traditional complementary DNA (cDNA) vectors utilising heterologous promoters. Viral vectors have proved, over the years, to be an effective means of gene delivery in vitro and in vivo, but the size of complete genomic loci precludes their use in most viral systems. One notable exception comprises the amplicon-type vectors based on human herpesviruses, such as the herpes simplex virus type I (HSV-1) amplicon vector. The large genome of HSV-1 (152 kb) confers upon HSV-1 amplicons a very large transgene capacity sufficient to accommodate approximately 95% of human genomic loci. The combination of the large transgene capacity, a broad range of cell tropism, and the ability to infect dividing and non-dividing cells makes HSV-1 amplicons an excellent vector system to develop for the delivery of large genomic loci. Here we review recent work which has shown that HSV-1 amplicons can be used for the delivery and expression of large genomic inserts >100 kb to cells in culture to rescue phenotypes in cellular models of genetic disease. We then discuss applications for high capacity HSV-1 amplicons in vivo and their potential to facilitate the use of large genomic inserts in gene therapy treatment regimes.

HSV Amplicon Vectors for Cancer Therapy

HSV amplicon vectors provide a unique tool in the armamentarium of weapons for treatment of cancer. Their large capacity (up to 150 kb) allows incorporation of multiple and large transgenes, including whole gene loci, as well as components of other viruses to control the fate of transgenes in the host cells. Means have been developed to achieve heritable transmission of transgenes in tumor cells by episomal replication or genomic integration. Therapeutic transgenes incorporated into amplicon vectors have included anti-angiogenic agents, immune enhancing proteins, prodrug activating enzymes, and apoptosis-inducing factors, as well as inhibitory RNAs for tumor-associated messages. Perks of this vector system include the ability to combine amplicon vectors with oncolytic HSV recombinant vectors to extend the therapeutic range and to target non-dividing as well as dividing tumor cells. Tumor vaccination is favored by the high infectivity of dendritic antigen-presenting cells with HSV vectors, and the vectors themselves appear to have intrinsic immune enhancing properties. Promoter manipulation can be used to target therapeutic gene expression to specific tumor cell types and to achieve drug regulated transgene expression. Further, amplicon vectors can be used to convert tumor cells into packaging cells for retrovirus and adeno-associated virus vectors, thus generating vectors on site. Amplicon vectors have also proven to be a versatile tool to explore imaging modalities to monitor gene delivery and tumor responses to therapeutic intervention.

Epigenetic silencing and tissue independent expression of a novel tetracycline inducible system in double‐transgenic pigs

The applicability of tightly regulated transgenesis in domesticated animals is severely hampered by the present lack of knowledge of regulatory mechanisms and the long generation intervals. To capitalize on the tightly controlled expression of mammalian genes made possible by using prokaryotic control elements, we have used a single-step transduction to introduce an autoregulative tetracycline-responsive bicistronic expression cassette (NTA) into transgenic pigs. Transgenic pigs carrying one NTA cassette showed a mosaic transgene expression restricted to single muscle fibers. In contrast, crossbred animals carrying two NTA cassettes with different transgenes, revealed a broad tissue-independent and tightly regulated expression of one cassette, but not of the other one. The expression pattern correlated inversely with the methylation status of the NTA transcription start sites indicating epigenetic silencing of one NTA cassette. This first approach on tetracycline regulated transgene expression in farm animals will be valuable for developing precisely controlled expression systems for transgenes in large animals relevant for biomedical and agricultural biotechnology.

Evidence for disease‐regulated transgene expression in the brain with use of lentiviral vectors

In this study we have developed and validated a novel approach of transgene regulation in the brain. By using lentiviral vectors that incorporate promoters of genes that are up-regulated during different pathological states, we were able to regulate transgene expression in accordance with the disease process. When using a glial fibrillary acidic protein promoter, efficient disease regulation in glial cells was achieved after an excitotoxic lesion or a 6-hydroxydopamine (6-OHDA) lesion. Transgene expression was physiologically regulated and displayed a dose-dependent increase depending on the severity of lesion. Efficient regulation was also achieved in neurons when using a preproenkephlin promoter in 6-OHDA-lesioned rats, allowing combined regulation and targeting. This disease-regulated approach allows control of transgene expression in the brain without the use of inducer molecules and without overexpression of transactivator proteins.

Role for Centromeric Heterochromatin and PML Nuclear Bodies in the Cellular Response to Foreign DNA

Nuclear spatial positioning plays an important role in the epigenetic regulation of eukaryotic gene expression. Here we show a role for nuclear spatial positioning in regulating episomal transgenes that are delivered by virus-like particles (VLPs). VLPs mediate the delivery of plasmid DNA (pDNA) to cell nuclei but lack viral factors involved in initiating and regulating transcription. By tracking single fluorescently labeled VLPs, coupled with luciferase reporter gene assays, we found that VLPs transported pDNA to cell nuclei efficiently but transgenes were immediately silenced by the cell. An investigation of the nuclear location of fluorescent VLPs revealed that the pDNAs were positioned next to centromeric heterochromatin. The activation of transcription by providing viral factors or inhibiting histone deacetylase activity resulted in the localization to euchromatin regions. Further, the activation of transcription induced the recruitment of PML nuclear bodies (PML-NBs) to the VLPs. This association did not play a role in regulating transgene expression, but PML protein was necessary for the inhibition of transgene expression with alpha interferon (IFN-alpha). These results support a model whereby cells can prevent foreign gene expression at two levels: by positioning transgenes next to centromeric heterochromatin or, if that is overcome, via the type I IFN response facilitated by PML-NB recruitment.

CaMKII Activation in the Entorhinal Cortex Disrupts Previously Encoded Spatial Memory

To investigate the role of the entorhinal cortex in memory at a molecular level, we developed transgenic mice in which transgene expression was inducible and limited to the superficial layers of the medial entorhinal cortex, pre- and parasubiculum. We found that expression of a constitutively active mutant form of CaMKII in these structures disrupted spatial memory formation. Immediate post-training activation of the transgene disrupted previously established memory while transgene activation 3 weeks following the training was ineffective. These results demonstrate that, similar to the hippocampus, the entorhinal cortex plays a time-limited role in spatial memory formation but is not a final cortical repository of long-term memory. Moreover, these results suggest that the indiscriminate activation of CaMKII is able to disrupt preexisting memories, possibly by altering the pattern of synaptic weight changes that are thought to form the basis of the memory trace.

Gene therapy of ovarian cancer with IFN-α-producing fibroblasts: comparison of constitutive and inducible vectors

Ovarian cancer represents a malignancy suitable for cell and gene therapy approaches owing to its containment within the peritoneal cavity, even at advanced tumor stages. As regulation of transgene expression would be preferable for conducting clinical trials for reasons of safety, we investigated whether intraperitoneal (i.p.) administration of retroviral vector-transduced fibroblasts encoding murine interferon-alpha (IFN-alpha) could have therapeutic activity, and compared its effect with the antitumor effects of fibroblasts producing IFN-alpha under a rapamycin analogue (AP21967)-inducible promoter. Human and murine fibroblasts were recruited into the solid component of transplantable ovarian cancer-grown i.p. in severe combined immunodeficiency mice. Multiple administrations of fibroblasts producing IFN-alpha in a constitutive manner showed therapeutic efficacy, leading to significant prolongation of survival in the majority of animals, associated with inhibition of tumor angiogenesis. Compared to cells transduced by the constitutive vector, fibroblasts transduced by the inducible vector released twofold higher IFN-alpha levels in vitro, following induction by AP21967, and production of the cytokine was under pharmacologic control both in vitro and in vivo. However, these cells elicited only modest therapeutic effects in vivo. Overall, these findings indicate that intracavitary IFN-alpha gene therapy using engineered fibroblasts requires sustained production of IFN-alpha to achieve durable antitumor effects.

Variable Recombination Efficiency in Responder Transgenes Activated by Cre Recombinase in the Vasculature

Cre recombinase has become a ubiquitous tool in transgenic strategies for regulation of transgene expression in a tissue-specific manner. We report analysis of two SM22alphaCre lines and their ability to mediate genomic recombination in five independent Cre-responsive transgenic lines. One of the SM22alphaCre lines developed was a tet-on system based on the reverse tetracycline transactivator. Our goal was to use this strategy to inhibit the Notch signaling pathway specifically in smooth muscle cells. Our responder transgenes contained a constitutively expressed marker gene (chloramphenicol acetyltransferase, CAT), flanked by loxP sites in direct orientation, upstream of Notch-related transgenes. We developed two dominant negative Notch transgenic responder lines activated by Cre-mediated DNA recombination. The first is the extracellular domain of human Jagged1, and the second is the extracellular domain of the human Notch2 receptor. Despite high expression of the marker gene in all responder lines, we found that Cre-mediated genomic recombination between these five lines was highly variable, ranging from 46 to 93% of individuals using an SM22alphaCre activating strain, or 8-58% of individuals using an inducible SM22alphartTACre. In all cases examined, detection of recombination by PCR correlated with expression of the transgene as determined by Western blot analysis. Our studies reflect the variability in recombination success based on the responder strain, presumably due to inaccessibility of the locus of integration of the responder allele.

Development of a methoxyfenozide‐responsive gene switch for applications in plants

The ecdysone receptor (EcR) has been used to develop gene switches for conditional regulation of transgene expression in plants and humans. All EcR-based gene switches developed to date for use in plants are monopartate and require micromolar concentrations of ligand for activation of the transgene; this has limited the use of these gene switches. We have developed a Choristoneura fumiferana ecdysone receptor (CfEcR)-based two-hybrid gene switch that works through the formation of a functional heterodimer between EcR and the retinoid X receptor (RXR) upon application of the chemical ligand methoxyfenozide. Methoxyfenozide is already registered for field use with an excellent safety profile, and it has potential as a gene switch ligand for applications in the field. The receptor constructs were prepared by fusing DEF domains (hinge region plus ligand-binding domain) of CfEcR to the GAL4 DNA-binding domain and EF domains (ligand-binding domain) of ultraspiracle from Choristoneura fumiferana (CfUSP) or RXR from Locusta migratoria (LmRXR), Mus musculus (MmRXR) or Homo sapiens (HsRXR) to the VP16 activation domain. These receptor constructs were tested for their ability to induce expression of the luciferase gene placed under the control of 5x GAL4 response elements and -46 35S minimal promoter in tobacco, corn and soybean protoplasts and in transgenic Arabidopsis and tobacco plants. By adopting the two-hybrid format, the sensitivity of the CfEcR gene switch has been improved from micromolar to nanomolar concentrations of methoxyfenozide. The sensitivity of the CfEcR + LmRXR two-hybrid switch was 25 to 625 times greater than the monopartate gene switch, depending on the plant species tested.

1016. Modulation of Transgene Expression: DNA Methylation Is, at Best, Only Partly Responsible

Many vectors are prone to waning transgene expression. To maintain expression one must first understand how inactivation occurs. Although SV40-derived vectors (rSV40s) are not susceptible to waning transgene expression, some transgenes (mainly of nonvertebrate origin, e.g., red fluorescent protein, RFP), express well when virus genomes are transfected but poorly on rSV40 transduction. Studying gene silencing in this system may provide useful insights into regulation of transgene expression.

959. Tumor Therapy Via Vector-Regulated Expression of Mutant Sodium Channel

Viral vectors provide a means to deliver therapeutic genes to tumors on site. In this study, we describe a novel tumor therapy employing a dominant-negative mutant form of a mammalian brain sodium channel, BNac1. This channel was delivered to tumor cells using an HSV amplicon vector in which gene expression is controlled by a tetracycline regulatory system (tet-on) with silencer elements. Channel activity, regulated transgene expression, cell death and the bystander effect were evaluated in glioma cells in culture. Upon infection and drug induction, the constitutively open channel is expressed leading to sodium and water influx into cells with swelling and death (Fig. 1). Gap junctions were demonstrated between tumor cells and mediated a bystander effect by ion influx whereby non-infected tumor cells adjacent to infected tumor cells were also killed. Growth and therapeutic response in culture and in vivo were monitored, in real-time, in experimental tumors expressing a recently characterized Gaussia luciferase that is far more sensitive than other luciferases currently in use.

369. Development of an Amplifiable Gene Expression System in Lentivirus Vectors

In certain applications of gene therapy, regulated (rather than constitutive) gene expression will be crucial for proper function or optimal effect of the delivered gene. Therefore, an important goal of gene therapy is to be able to deliver genes so that they express in a pattern that recapitulates the expression of an endogenous cellular gene. Additionally, regulating transgene expression will be an important component of efforts for reducing risks of adverse advents associated with gene therapy with integrating vectors. Although tissue-specific promoters confer selectivity, in a vector- based system, their activity may be too weak to mediate sufficient levels of gene expression to achieve therapeutic efficacy in vivo. Additionally, amplification of a specific, but weak, signal would be beneficial to some applications, such as imaging. Iyer et al. (PNAS, 2001) described a two-step transcriptional activator (TSTA) system to amplify gene expression while preserving tissue specificity using adenoviral vectors. In this system, the tissue-specific promoter drives expression of a potent synthetic transcription activator (the yeast GAL4 DNA binding domain fused to the activation domain of the HSV-1 VP16 activator), which in turn activates a GAL4-responsive reporter. We have developed lentivirus-based vectors using this 2-tiered system with the constitutive but weak PGK housekeeping gene promoter and the human insulin promoter and have tested them in cell lines and primary cells. Compared to a vector carrying only the PGK promoter to drive eGFP expression, the PGK amplifiable- vector amplified eGFP expression from 2–11 fold, depending on the cell type. Vectors carrying the insulin promoter did not express in non-β cell lines (HT29, HEK293), but expressed in Min6 cells, a murine insulinoma cell line, indicating that the insulin promoter was capable of conferring cell specificity. Additionally, the insulin amplifiable-vector was able to amplify expression 4.6 times in these cells. In primary murine islets, no gene expression was detected from the vector carrying only the insulin promoter, but low eGFP expression was detected from the insulin-promoter amplifiable vector. Thus, we have demonstrated that it is possible to build lentivirus vectors carrying an amplifiable gene expression system that retains tissue specificity. These vectors will be useful in gene expression studies that require a detectable signal and tissue specificity.

Analysis of Tissue-Specific Expression of Human Type II Collagen cDNA Driven by Different Sizes of the Upstream Region of the β-Casein Promoter

To investigate the ability of 1.8 kb or 3.1 kb bovine beta-casein promoter sequences for the expression regulation of transgene in vivo, transgenic mice were produced with human type II collagen gene fused to 1.8 kb and 3.1 kb of bovine beta-casein promoter by DNA microinjection. Five and three transgenic founder mice were produced using transgene constructs with 1.8 kb and 3.1 kb of bovine beta-casein promoters respectively. Founder mice were outbred with the wild type to produce F1 and F2 progenies. Total RNAs were extracted from four tissues (mammary gland, liver, kidney, and muscle) of female F1 transgenic mice of each transgenic line following parturition. RT-PCR and Northern blot analysis revealed that the expression level of transgene was variable among the transgenic lines, but transgenic mice containing 1.8 kb of promoter sequences exhibited more leaky expression of transgene in other tissues compared to those with 3.1 kb promoter. Moreover, Western blot analysis of transgenic mouse milk showed that human type II collagen proteins secreted into the milk of lactating transgenic mice contained 1.8 kb and 3.1 kb of bovine beta-casein promoter. These results suggest that promoter sequences of 3.1 kb bovine beta-casein gene can be used for induction of mammary gland-specific expression of transgenes in transgenic animals.

Regulatable Gutless Adenovirus Vectors Sustain Inducible Transgene Expression in the Brain in the Presence of an Immune Response against Adenoviruses

In view of recent serious adverse events and advances in gene therapy technologies, the use of regulatable expression systems is becoming recognized as indispensable adjuncts to successful clinical gene therapy. In the present work we optimized high-capacity adenoviral (HC-Ad) vectors encoding the novel tetracycline-dependent (TetOn)-regulatory elements for efficient and regulatable gene expression in the rat brain in vivo. We constructed two HC-Ad vectors encoding beta-galactosidase (beta-gal) driven by a TetOn system containing the rtTAS(s)M2 transactivator and the tTS(Kid) repressor under the control of the murine cytomegalovirus (mCMV) (HC-Ad-mTetON-beta-Gal) or the human CMV (hCMV) promoter (HC-Ad-hTetON-beta-Gal). Expression was tightly regulatable by doxycycline (Dox), reaching maximum expression in vivo at 6 days and returning to basal levels at 10 days following the addition or removal of Dox, respectively. Both vectors achieved higher transgene expression levels compared to the expression from vectors encoding the constitutive mCMV or hCMV promoter. HC-Ad-mTetON-beta-Gal yielded the highest transgene expression levels and expressed in both neurons and astrocytes. Antivector immune responses continue to limit the clinical use of vectors. We thus tested the inducibility and longevity of HC-Ad-mediated transgene expression in the brain of rats immunized against adenovirus by prior intradermal injections of RAds. Regulated transgene expression from HC-Ad-mTetON-beta-Gal remained active even in the presence of a significant systemic immune response. Therefore, these vectors display two coveted characteristics of clinically useful vectors, namely their regulation and effectiveness even in the presence of prior immunization against adenovirus.