Regulation Of Transgene Expression Research Articles

Bone morphogenetic protein gene therapy.

A retrospective analysis of previous BMP gene therapy and general gene therapy publications. To present the potential role of BMP gene therapy for the induction of osteogenesis and spinal fusion. A variety of viral and non-viral techniques have been utilized to insert foreign transgenes into cells, both in vivo and in vitro. These techniques are now being used to transduce cells with a BMP gene to express significant amounts of BMP. This secreted BMP can subsequently stimulate osteogenesis in a variety of locations, including in the paraspinal regions. A retrospective analysis of the literature. Direct and ex vivo BMP gene therapy has been shown to successfully promote bone healing and regeneration in a variety of animal models. Long-term and regulated transgene expression are clear advantages of BMP gene delivery, compared to direct BMP application. To date, BMP gene delivery with adenoviral vectors have been the most effective approach for stimulating bone induction in vivo. Although BMP gene therapy techniques have significant potential for the treatment of spine pathology, further preclinical and clinical research and development are required before this technology will have direct clinical applications.

Inducible control of tissue-specific transgene expression in Xenopustropicalis transgenic lines

Analysis of gene function in vertebrates is facilitated by gain-of-function studies, such as injection of synthetic mRNA in amphibian embryos. This approach is hampered by lack of spatial and temporal control of expression of the introduced gene product. An additional level of control is obtained by nuclear-transfer-mediated transgenesis, but functional analyses are complicated by variability and background abnormalities in primary transgenic embryos. The GAL4/UAS system permits establishment of stable lines and elimination of nuclear-transfer-associated abnormalities, through generation of separate UAS-‘effector’ and GAL4 ‘transactivator’ transgenic lines. When the GAL4 DNA-binding domain is combined with a steroid hormone ligand-binding domain, this system allows full temporal regulation of transgene expression by introduction of an exogenous steroid analogue, the progesterone antagonist RU486. We show here that by crossing stable Xenopus tropicalis transgenic lines, one bearing a UAS-enhanced cyan fluorescent protein (ECFP) reporter construct, and the other with a GAL4–progesterone receptor fusion driven by a retina-specific promoter, reporter expression in the resulting embryos can be induced with RU486 in a tissue-specific manner. These results suggest that the inducible binary system, in which the target gene expression can be controlled in a stage- and tissue-specific pattern, should be readily applicable for gene function studies at all stages of development.

Distinct Enhancers Regulate Skeletal and Cardiac Muscle-Specific Expression Programs of the Cardiac α-Actin Gene in Xenopus Embryos

During vertebrate embryonic development, cardiac and skeletal muscle originates from distinct precursor populations. Despite the profound structural and functional differences in the striated muscle tissue they eventually form, such progenitors share many features such as components of contractile apparatus. In vertebrate embryos, the α-cardiac actin gene encodes a major component of the myofibril in both skeletal and cardiac muscle. Here, we show that expression of Xenopus cardiac α-actin in the myotomes and developing heart tube of the tadpole requires distinct enhancers within its proximal promoter. Using transgenic embryos, we find that mutations in the promoter-proximal CArG box and 5 bp downstream of it specifically eliminate expression of a GFP transgene within the developing heart, while high levels of expression in somitic muscle are maintained. This sequence is insufficient on its own to limit expression solely to the myocardium, such restriction requiring multiple elements within the proximal promoter. Two additional enhancers are active in skeletal muscle of the embryo, either one of which has to interact with the proximal CArG box for correct expression to be established. Transgenic reporters containing multimerised copies of CArG box 1 faithfully detect most sites of SRF expression in the developing embryo as do equivalent reporters containing the SRF binding site from the c-fos promoter. Significantly, while these motifs possess a different A/T core within the CC(A/T)6GG consensus and show no similarity in flanking sequence, each can interact with a myotome-specific distal enhancer of cardiac α-actin promoter, to confer appropriate cardiac α-actin-specific regulation of transgene expression. Together, these results suggest that the role of CArG box 1 in the cardiac α-actin gene promoter is to act solely as a high-affinity SRF binding site.

1297. Regulated Transgene Expression in B-Lymphocytes from Lentiviral Vectors Carrying the Immunoglobulin Heavy Chain Enhancer with Matrix Attachment Regions

1297. Regulated Transgene Expression in B-Lymphocytes from Lentiviral Vectors Carrying the Immunoglobulin Heavy Chain Enhancer with Matrix Attachment Regions

407. Development of a Novel Tetracycline-Inducible Lentiviral Vector System for the Regulation of Transgene Expression

407. Development of a Novel Tetracycline-Inducible Lentiviral Vector System for the Regulation of Transgene Expression

506. Systemic and Localized Reversible Regulation of Transgene Expression by Tetracycline with tetR-Mediated Transcription Tepression Switch

506. Systemic and Localized Reversible Regulation of Transgene Expression by Tetracycline with tetR-Mediated Transcription Tepression Switch

415. Single Adenovirus Vector Containing Both rtTA and tTS Expression Cassettes Provides Tight Regulation of Transgene Expression in a Positive Manner

415. Single Adenovirus Vector Containing Both rtTA and tTS Expression Cassettes Provides Tight Regulation of Transgene Expression in a Positive Manner

79. Robust and Efficient Regulation of Transgene Expression In Vivo by Improved Lentiviral Vectors

79. Robust and Efficient Regulation of Transgene Expression In Vivo by Improved Lentiviral Vectors

93. Characterization of Regulated Transgene Expression from Inducible Lentivirus Vectors in Primary Cells in Culture and In Vivo

93. Characterization of Regulated Transgene Expression from Inducible Lentivirus Vectors in Primary Cells in Culture and In Vivo

The tet-off system is more effective than the tet-on system for regulating transgene expression in a single adenovirus vector.

Control of transgene expression in mammalian cells is desirable for gene therapy and the study of gene function in basic research. This study evaluates the functionality of single adenovirus (Ad) vectors containing a tetracycline-controllable expression system (tet-off or tet-on system). An Ad-mediated binary transgene expression system was generated containing a tet-off or a tet-on system, which introduced the gene of interest with a tetracycline-regulatable promoter and the tetracycline-responsive transcriptional activator gene into the E1 and E3 deletion regions, respectively. The functionality of the Ad-mediated tet-off and tet-on systems was compared under various conditions in vitro and in vivo. The Ad vector containing the tet-off system provided negative control of gene expression ranging from 20- to 500-fold, depending on the cell type and condition. In contrast, the Ad vector containing the tet-on system increased gene expression by only 2- to 28-fold and required about two-log orders higher concentration of an inducer (doxycycline) to switch on the gene expression, compared with the Ad vector containing the tet-off system. Ad vectors containing the tet-off system are a better choice for regulated gene expression than Ad vectors containing the tet-on system. Single Ad vectors containing the tetracycline-controllable expression system will greatly facilitate in vitro and in vivo analyses of gene function and may be useful for gene therapy.

Robust and efficient regulation of transgene expression in vivo by improved tetracycline-dependent lentiviral vectors.

We developed a panel of lentiviral vectors that displayed tetracycline-regulated transgene expression over two orders of magnitude in bulk, non-selected populations of transduced cells in vitro and in vivo. The robust expression and homogeneous response indicated that most transduced vector genomes were transcription competent and responsive to regulation, providing the lentiviral vector with a novel competitive advantage for gene transfer. After ex vivo transduction and transplantation of cord blood CD34+ cells into NOD/SCID mice, reporter gene expression could be switched "on" and "off" in human hematopoietic cells in vivo for prolonged times, proving integration of the regulated expression system into long-term repopulating cells. By vector injection into established tumor grafts, we achieved efficient delivery and quantitative regulation of transgene expression in vivo. By these approaches, gene function studies can now be performed in in vivo models of human hematopoiesis and cancer. In the future, regulated lentiviral vectors will improve the safety and efficacy of gene therapy.

Globin gene transfer for the treatment of severe hemoglobinopathies: a paradigm for stem cell-based gene therapy.

The prospect of treating blood disorders with genetically modified stem cells is highly promising. This therapeutic approach, however, raises a number of fundamental biological questions, spanning several research fields. Further investigation is required to better understand how to isolate and efficiently transduce hematopoietic stem cells (HSCs), while preserving optimal homing and self-renewing properties; how to design safe vectors permitting controlled expression of the transgene products; and how to promote host repopulation by engrafted HSCs. This article addresses basic issues in stem cell-based gene therapy from the perspective of regulating transgene expression, taking globin gene transfer for the treatment of severe hemoglobinopathies as a paradigm.

Chloroplast targeting signal of a rice rbcS gene enhances transgene expression

Previously, we reported that the presence of therbcS transit peptide (TP) sequence as a plastid-targetingsignal led to greatly enhanced accumulation of transgene products in transgenicrice plants. To study the mechanisms underlying the regulation of transgeneexpression by the TP sequence, we mutated the sequence(mTP) so that it did not localize fused heterologousproteins into plastids, while it retained a nearly identical nucleotidesequenceto its wild type. mTP was fused to a synthetic gene(sgfp) encoding a modified form of the green fluorescentprotein (sGFP) and linked to the rice rbcS promoter,generating an expression vector, rbcS-mTP-sgfp. To compareexpression levels in transgenic plants, two expression vectors wereconstructed:rbcS-sgfp for untargeted expression andrbcS-TP-sgfp for chloroplast-targeted expression. Severaltransgenic plants were produced for each construct by theAgrobacterium-mediated method. Confocal microscopicanalyses demonstrated that GFP fluorescence in therbcS-TP-sgfp-transformed lines was specifically localizedwithin chloroplasts, whereas fluorescence was detected within cytoplasm andnucleoplasm in the rbcS-mTP-sgfp- andrbcS-sgfp-transformed lines. Northern blot analysisindicated that mRNA levels were very different, depending on the type ofTP sequence employed. Transcript levels ofrbcS-TP-sgfp-transformed lines were 10 fold higher thanthose of either rbcS-mTP-sgfp- orrbcS-sgfp-transformed lines, whereas the levels of thelasttwo were comparable. Levels of sGFP accumulation were also correspondinglyhigher in rbcS-TP-sgfp-transformed lines than in the othertwo lines, as examined by immunoblot analyses. Thus, increased levels of sGFPaccumulation appeared to reflect increased mRNA levels; this was due to thepresence of the functional rbcS transit peptide. Theseresults suggest that a functional rbcS transit peptideenhances transgene expression in addition to targeting the gene products intochloroplasts.

Regulated and prolonged expression of mIFN(alpha) in immunocompetent mice mediated by a helper-dependent adenovirus vector.

A major goal in gene therapy is to develop efficient gene transfer protocols that allow tissue-specific, long-term and tightly regulated expression of the desired transgene. This objective is becoming more attainable through the co-evolution of gene transfer vectors and regulation systems. The ideal vector should efficiently transduce non-dividing cells with minimal toxicity, thus endowing the system with persistent transgene expression. The helper-dependent adenovirus vectors meet these requirements, as demonstrated in various studies in the literature. The most promising regulation system is the tet-on system, which has low basal transcriptional activity and high inducibility. To explore the regulated transgene expression in the context of a helper-dependent vector, we constructed the HD-TET-IFN vector, containing the mIFNα gene under the control of the tetracycline inducible transactivator rtTA2s-S2. Mice injected with HD-TET-IFN showed high levels of serum mIFNα only upon transcriptional activation. The transgene expression was reinducible to the same high level up to 3 months p.i., and the amount of expressed cytokine could be regulated by dosing doxycycline. Transcriptional activation of mIFNα induced by doxycycline resulted in prolonged survival and reduced liver damage in HD-TET-IFN-injected mice challenged with a lethal dose of coronavirus. Activation of antiviral genes mediated by doxycycline-dependent mIFNα expression was also observed at low HD-TET-IFN doses. The possibility of controlling gene expression by the combination of HD vectors and the latest tet-on transactivator also holds promise for studying gene function in other animal models.

Retroviral vectors for the transduction of autoregulated, bidirectional expression cassettes.

Regulated transgene expression is increasingly used in research but is also needed for certain therapies. Regulatory systems are usually composed of two expression units, one bearing the gene of interest under control of a regulatable promoter and the other, a constitutively expressed transactivator that modulates the activity of the regulatable promoter. Because the cotransfer of two independent elements is not efficient in primary cells, single transduction step vectors conferring regulatable gene expression cassettes would be helpful. We have developed retroviral vectors containing an autoregulatory bidirectional expression cassette that encodes all components necessary for regulated expression of a gene of interest. The influence of the orientation of the reporter gene with respect to the viral long terminal repeat (LTR) and the effect of transcriptionally inactive LTRs were investigated using mouse leukemia virus (MLV) and self-inactivating (SIN)-based retroviral vectors. Strict regulation was observed when the reporter was inserted in antisense orientation with respect to the LTR, whereas a sense arrangement of the reporter resulted in a loss of regulation capacity. Expression and regulation of the antisense-orientated reporter gene were homogenous in infected cell pools and investigated cell clones. Long-term observations of infected cells over a period of 30 passages revealed stable expression and regulation. These autoregulated, bidirectional retroviral vectors combine the advantages of single-step transduction with strict regulation of the gene of interest in the infected target cells.

A complex adenovirus vector that delivers FASL-GFP with combined prostate-specific and tetracycline-regulated expression.

Cell-type-restricted transgene expression delivered by adenovirus vectors is highly desirable for gene therapy of cancer, as it can limit cytotoxic gene expression to tumor cells. However, many tumor- and tissue-specific promoters are weaker than the constitutively active promoters and are thus less effective. To combine cell-type specificity with high-level regulated transgene expression, we have developed a complex adenoviral vector. We have placed the tetracycline transactivator gene under the control of a prostate-specific ARR2PB promoter, and a mouse Tnfsf6 (encoding FASL)-GFP fusion gene under the control of the tetracycline responsive promoter. We have incorporated both expression cassettes into a single construct. We show that FASL-GFP expression from this vector is essentially restricted to prostate cancer cells, in which it can be regulated by doxycycline. Higher levels of prostate-specific FASL-GFP expression were generated by this approach than by driving the FASL-GFP expression directly with ARR2PB. More FASL-GFP expression correlated with greater induction of apoptosis in prostate cancer LNCaP cells. Mouse studies confirmed that systemic delivery of both the prostate-specific and the prostate-specific/tet-regulated vectors was well tolerated at doses that were lethal for FASL-GFP vector with CMV promoter. This strategy should be able to improve the safety and efficacy of cancer gene therapy using other cytotoxic genes as well.

Toward gene therapy for disorders of globin synthesis

Inherited disorders of hemoglobin remain desirable targets for genetically based therapies. That stem cell replacement reverses the phenotype of both thalassemia and sickle cell anemia has been well established through allogeneic bone marrow transplantation studies, yet significant toxicities and finite donor availability limit this approach to a minority of affected individuals. Genetically based strategies that have as their goal addition of a normal copy of the human β-globin gene along with key regulatory sequences to autologous hematopoietic stem cells represent a viable alternative to allogeneic transplantation, but this approach has been impeded by formidable obstacles over the last decade. Large animal models have become the standard for the development of clinically relevant gene addition strategies, and significant progress in the techniques used to deliver potentially therapeutic genes has been achieved. The clinical application of such strategies may be close at hand, at least for disorders in which modest level, constitutive expression is sufficient to correct the phenotype. For the thalassemias and hemoglobinopathies, complex, regulated, lineage specific expression of the β-globin gene at relatively high levels will be required. The discovery of the β-globin locus control region renewed interest in the thalassemias and sickle cell anemia as targets for gene transfer, but difficulties in attaining high-titer vectors along with a tendency toward rearrangement when segments of the locus control region (LCR) were incorporated into retroviral vectors stalled further progress. Recent advances in vector construction have circumvented this problem and others limiting both gene transfer efficiency and regulation of transgene expression, offering new hope for clinical application.

Efficient and ligand-dependent regulated erythropoietin production by naked DNA injection and in vivo electroporation

The development of an in vivo gene transfer approach to deliver physiologic levels of recombinant proteins to the systemic circulation would represent a significant advance in the treatment of protein deficiencies-disorders. However, the ability to regulate transgene expression will become paramount for safety and efficacy in gene transfer therapy. We have described the construction of an efficient and ligand-dependently regulated erythropoietin (EPO) production system using naked plasmid and in vivo electroporation. Two plasmids, one encoding the chimeric GeneSwitch protein and the other encoding an inducible transgene for human EPO, were developed. Modulation of the level of secretion of EPO into the serum was achieved by intraperitoneal administration of mifepristone (MFP). Rats were divided into 4 groups: one group received EPO plasmid with MFP for 30 days, a second group received with EPO plasmid MFP for 9 days, a third group received EPO plasmid without MFP, and a fourth group received control plasmid. A pair of electrodes was inserted into the muscle of the right thigh and 100 μg of each plasmid was injected. In vivo electrporation (8 times at 100 V for 50 milliseconds) was performed. The presence of vector-derived EPO mRNA was detected by reverse transcriptase-polymerase chain reaction only in the EPO and MFP(+) group. The hematocrit levels increased continuously from the preinjection level of 42.7% to 53.8% on day 28 in the EPO and MFP(+) group. The serum EPO levels increased only in the EPO and MFP(+) group. There was no significant change in hematocrit levels and EPO levels in the EPO and MFP(−) group. These results demonstrate that EPO gene transfer with the GeneSwitch system by in vivo electroporation is a useful procedure for efficient and drug-dependent regulated delivery of EPO. © 2001 by the National Kidney Foundation, Inc.

Characteristics of adenovirus-mediated tetracycline-controllable expression system

The combination of recombinant adenovirus (Ad) vectors and the tetracycline-controllable expression system is clearly an advantage in gene therapy and gene transfer experiment. In this study, we examined the characteristics of Ad vectors containing the tet-off or tet-on system. The Ad vector containing the tet-off system showed tightly regulatable transgene expression even at low MOI (multiplicity of infection). In contrast, regulation of gene expression by the Ad vector containing the tet-on system was not tight at low MOI, while it showed moderate regulation at high MOI (MOI=100). The Ad vector-mediated tet-on system showed lower inducible and higher background (basal) luciferase production than that of the Ad vector-mediated tet-off system. Moreover, the former system required a concentration of doxycycline, a derivative of tetracycline, approx. 2–3 log orders higher than that of the latter system to switch the luciferase expression. A combination of the vector containing the tet-on system and the vector containing the tetracycline-controlled transcriptional silencer (tTS) gene reduced the background luciferase production and improved regulation. These results suggest that the Ad vector containing the tet-off system is considered to be functionally superior to the vector containing the tet-on system. Care should be taken regarding regulation (especially lower inducibility and higher background), which is decreased in the Ad vector-mediated tet-on system in comparison with the tet-off system. The Ad vector containing the tetracycline-controllable expression system should offer a powerful tool for gene therapy and gene transfer experimentation.

Plant selectable markers and reporter genes

In recent years, considerable progress has been made in genetic engineering of various plant species, both agronomically important crops as well as model plants. The bases of this progress were, in addition to efficient transformation methods, the design of appropriate signals regulating transgene expression and the use of selection marker or reporter genes. In most cases, a gene of interest is introduced into plants in association with a selectable marker gene (nptII, hpt, acc3, aadA, bar, pat). Recovery of a transgenic plant is, therefore, facilitated by selection of putative transformants on a medium containing a selection agent, such as antibiotic (nptII, hpt, acc3, aadA), antimetabolite (dhfr), herbicide (bar, pat), etc. On the other hand, use of reporter genes (cat, lacZ, uidA, luc, gfp) allows not only to distinguish transformed and non-transformed plants, but first of all to study regulation of different cellular processes. In particular, by employing vital markers (Luc, GFP) gene expression, protein localization and intracellular protein traffic can be now observed in situ, without the need of destroying plant.