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At least 19 recordsLinked to original sources
PubMed2007

Novel gene switches.

Controlling gene activity in space and time represents a cornerstone technology in gene and cell therapeutic applications, bioengineering, drug discovery as well as fundamental and applied research. This chapter provides a comprehensive overview of the different approaches for regulating gene activity and product protein formation at different biosynthetic levels, from genomic rearrangements over transcription and translation control to strategies for engineering inducible secretion and protein activity with a focus on the development during the past 2 years. Recent advances in designing second-generation gene switches, based on novel inducer administration routes (gas phase) as well as on the combination of heterologous switches with endogenous signals, will be complemented by an overview of the emerging field of mammalian synthetic biology, which enables the design of complex synthetic and semisynthetic gene networks. This article will conclude with an overview of how the different gene switches have been applied in gene therapy studies, bioengineering and drug discovery.

Animals↗
PubMed1982

Nucleotide sequence and properties of the murine gamma 3 immunoglobulin heavy chain gene switch region: implications for successive C gamma gene switching.

During B lymphocyte differentiation, immunoglobulin heavy chain constant region (CH) genes undergo a unique series of DNA recombination events culminating in the CH class switch. CH switch (S) regions are located 2 kb 5' of each CH gene except delta (i.e. mu, gamma 3, gamma 1, gamma 2b, gamma 2a, epsilon and alpha). We describe the structural features of the gamma 3 switch region. Hybridization experiments show that S gamma 3 has remarkable homology to both S mu and other S gamma regions while S mu possesses limited homology to the other S gamma sequences. However, S mu possesses extensive sequence homology with S epsilon and S alpha. The nucleotide sequence of S gamma 3 reveals higher densities of S mu repetitive sequences (GAGCT and GGGGT) and another S region common sequence (YAGGTTG) than observed for S gamma 1, S gamma 2b or S gamma 2a. In addition, the conservation of S mu like repetitive sequences in S gamma regions is correlated with the 5' leads to 3' gamma gene order (i.e. S gamma 3 greater than S gamma 1 greater than S gamma 2b greater than S gamma 2a). A model is presented which suggests that the unique features of S gamma 3 may allow for successive switches from C mu to any C gamma gene.

Animals↗
PubMed1982

On immunoglobulin heavy chain gene switching: two gamma 2b genes are rearranged via switch sequences in MPC-11 cells but only one is expressed.

During B lymphocytes differentiation, switches in the expression of heavy chain immunoglobulin constant region (CH) genes occur by a novel DNA recombination mechanism. We have investigated the requirements of the CH gene switch by characterizing two rearranged gamma 2b genes from a gamma 2b producing mouse myeloma (MPC-11). One of the two gamma 2b genes is present in 2-3 copies per cell (gamma 2b strong hybridizer) while the other is present in approximately 1 copy per cell (gamma 2b weak hybridizer). Genomic clones of the gamma 2b strongly hybridizing gene indicate that this is an abortive switch event between the S gamma 3 and S gamma 2b regions. However, clones of the gamma 2b weakly hybridizing gene suggest a functional rearrangement due to the presence of VH, JH and S mu sequences. The switch-recombination sites of these rearranged gamma 2b genes and those of other CH genes show a high degree of preference for the sequence AGGTTG 5' of either the S mu donor site or the appropriate CH S acceptor site. AGGTTG and its analogs are rare in the S mu region, are somewhat prevalent in s alpha and in the case of S mu are found 5' of a tandemly repeated DNA sequence (GAGCT, GGGGT) comprising most of S mu.

Animals↗
PubMed1990

A set of human putative lymphocyte G0/G1 switch genes includes genes homologous to rodent cytokine and zinc finger protein-encoding genes.

Lymphocyte G0/G1 switch genes (G0S genes) are potential oncogenes and may regulate, be regulated by, or be coordinately regulated with, latent lymphotropic viruses. To identify these genes, a cDNA library was prepared from blood mononuclear cells that had been cultured for 2 hr with a T-cell mitogen (lectin) and cycloheximide. Eight differentially hybridizing recombinants were characterized by RNA and DNA blotting and sequencing. One cDNA (G0S7) corresponded to the oncogene c-fos. Another cDNA (G0S19) was homologous (70%) to a cDNA encoding a murine inhibitor of stem cell proliferation (the cytokine MIP1 alpha) and, less closely, to other members of the "small inducible" secreted protein-encoding gene family. Whereas cDNA hybridization to genomic DNA blots indicated a small subfamily of G0S19 genes, simple patterns of bands indicated that most cDNAs, including G0S30 cDNA, corresponded to single-copy genes. The 3' noncoding sequence of G0S30 cDNA was homologous (87-89%) to the 3' noncoding sequences of certain rodent genes (NGFI-A, Krox24, EGR1) that encode zinc finger proteins (putative transcriptional regulators). This degree of evolutionary conservation suggests an important function for the 3' noncoding region. The 3' noncoding regions of some cDNAs contained the TTATTTAT (mRNA destabilization) element. The corresponding RNAs each formed doublets in agarose gels. Previous studies of c-fos RNA from HeLa cells indicate that this is due to cycloheximide-dependent stabilization of poly(A) tails. Our results reveal the power of cycloheximide enrichment in isolating what would appear to be significant low-abundance mRNAs.

Base Sequence↗
PubMed1992

The protein sequence and some intron positions are conserved between the switching gene swi10 of Schizosaccharomyces pombe and the human excision repair gene ERCC1.

The switching gene swi10+ has a function in mating-type switching as well as in the repair of radiation damages. We have cloned the genomic swi10+ gene by functional complementation of the switching defect of the swi10-154 mutant. The swi10+ gene is not essential for viability. The DNA sequence revealed an open reading frame of 759 nucleotides interrupted by three introns of 127, 52 and 60 bp, respectively. The positions of intron I as well as of intron III of swi10 are evolutionary conserved in comparison to the introns III and IV of the human ERCC1 gene. The analysis of cDNA clones isolated by PCR amplification confirmed the structure of the swi10 gene. The putative Swi10 protein has homologies to the human and mouse ERCC1 protein, to Rad10 of Saccharomyces cerevisiae and to parts of UvrA and UvrC of E. coli. All these proteins are essential components for excision repair of damaged DNA. The Swi10 protein contains a putative DNA binding domain previously found in other proteins. Northern blot experiments and the analyses of cDNA clones indicate that intron I of the swi10 gene is not efficiently spliced.

Amino Acid Sequence↗
PubMed2003

Pharmacogenetic rescue in time and space of the rutabaga memory impairment by using Gene-Switch.

The GAL4-based Gene-Switch system has been engineered to regulate transgene expression in Drosophila in both time and space. We constructed a Gene-Switch transgene in which Gene-Switch expression is restricted spatially by a defined mushroom body enhancer. This system allows Gene-Switch to be active only in the mushroom bodies and only on administration of the pharmacological Gene-Switch ligand RU486. This line was used to drive the expression of a rutabaga cDNA in otherwise rutabaga mutant flies. Induction of the rutabaga cDNA in the mushroom bodies only during adulthood, or during adulthood along with the larval and pupal developmental stages, corrects the olfactory memory impairment found in rutabaga mutants. Induction of the cDNA only during the larval and pupal stages was inconsequential to performance in olfactory memory tasks. These data indicate that normal rutabaga function must be expressed in adulthood for normal memory and conclusively delimit the time and space expression requirements for correcting the rutabaga memory impairment. Such combined pharmacogenetic regulation of transgene expression now allows this time and space dissection to be made for other behavioral mutants.

Adenylyl Cyclases↗
PubMed2006

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.

Animals↗
PubMed2000

Reporter constructs showing proper globin gene switching in cultured erythroid cell lines.

Recent advances in the study of globin gene switching in the context of complete gene locus have contributed greatly to our understanding of developmental regulation mechanism of globin gene expression. However, it is not clear yet whether the cluster is sufficient in proper gene switching when the globin genes are replaced with conventional reporter genes. Furthermore, even though erythroid-specific and ubiquitous transcription factors involved in erythroid-specific globin gene expression have been characterized and some plausible globin gene switching models have been suggested, any specific factor directly involved in globin gene switching is not yet identified. In this study, as an effort to further understand globin switching mechanism and to identify globin switching factors, we constructed reporter vectors by juxtaposing several putative regulatory elements in human beta-globin locus to conventional reporter genes and analyzed their stage-specific expression in erythroid cell lines. At the end, we demonstrated that gammabeta-type constructs, in which both gamma-type and beta-type globin reporter genes were linked in cis below beta-globin locus control region (LCR), show proper stage-specific reporter gene expression in erythroid cell lines and also recapitulate globin switching in cell hybrids.

Animals↗
PubMed2001

The regulatory network controlling beta-globin gene switching.

The human globin gene cluster, which represents a prototypical eukaryotic multigene locus, has been investigated for more than two decades and is classic model for coordinate control of tissue-specific gene expression. It is well known that globin gene expression is restricted to specific tissues and that globin genes are sequentially switched on during development. What intricate regulatory mechanisms account for tissue-specific transcriptional control of globin gene expression? Previous studies have focused on the interactions of trans-acting factors and cis-acting elements including the locus control region (LCR), which is considered a potent enhancer in globin gene switching. More recent studies have not only focused on the local DNA regulatory elements but also on remodelling of chromatin and transcription at the globin gene cluster within the native genomic context. Moreover, several studies have presented extensive data that address whether the LCR is required to open the chromatin. Although there is increased insight into the regulation of the beta-globin gene switching, many aspects relating to the developmental activation of distinct globin genes remain elusive.

Animals↗
PubMed2002

Comparative study and identification of potent eukaryotic transcriptional repressors in gene switch systems.

In mammalian cells, proper gene regulation is achieved by the complex interplay of transcription factors that activate or repress gene expression by binding to the regulatory regions of target promoters. While transcriptional activators have been extensively characterised and classified into functional groups, relatively little is known about the comparative strength and cell type-specificity of transcriptional repressors. Here, we have compared the ability of a series of eukaryotic repression domains to silence basal and activated transcription. A series of the most potent repression domains was further tested in the context of a gene therapy gene-switch system in various cell types. The results indicate that the analysed repression domains exert varying silencing activities in different promoter contexts. Furthermore, their potential for gene silencing varies also depending on the cellular context. When multimerised within one chimeric repressor protein, particular combinations of repressor domains were found to display synergistic repressing effects and efficient repression in a panel of cell lines. This approach thus allowed the identification of transcriptional repressors that are both potent and versatile in terms of cellular specificity as a basis for gene switch systems.

Cell Line↗
PubMed1997

Globin gene switching in transgenic mice carrying HS2-globin gene constructs.

We have examined the pattern of human globin gene switching in transgenic mice containing three different gamma and beta gene constructs (HS2G gamma A gamma delta beta, HS2A gamma beta neo, and HS2A gamma en beta) and compared the results with previously described transgenics (HS2A gamma beta, HS2G gamma A gamma-117 delta beta, and LCR epsilon G gamma A gamma delta beta). Developmental regulation was observed in all cases with identical patterns in lines bearing the same construct. Three different patterns of switching were observed: LCR epsilon G gamma A gamma delta beta and HS2A gamma beta neo mice switched rapidly, HS2G gamma A gamma delta beta and HS2G gamma A gamma-117 delta beta at an intermediate rate, and HS2A gamma beta and HS2A gamma en beta mice showed delayed switching, with a plateau in late fetal-early neonatal life and readily detectable levels of gamma mRNA in adults. No difference was observed in the time of switching of the HS2G gamma A gamma delta beta mice compared with those with the A gamma-117 hereditary persistence of fetal hemoglobin mutation, but adult levels of gamma mRNA were significantly higher (approximately 5%) in lines carrying the mutation than in those without (approximately 1%). Reversion to the rapid switch of the LCR epsilon G gamma A gamma delta beta mice was observed in three lines with the HS2A gamma beta neo construct in which expression of the tk-neo gene was approximately equal to that of the globin genes. The inclusion of the A gamma enhancer in HS2A gamma beta mice did not alter the pattern of switching, or reduce the relatively high levels of gamma mRNA in these lines. However, unlike other HS2 mice, the combination of HS2 and the A gamma enhancer resulted in copy number-dependent expression in HS2A gamma en beta lines, with intrauterine death at approximately 12.5 days gestation at high copy numbers. These results demonstrate that numerous elements throughout the beta globin gene cluster interact to produce the correct pattern of developmental regulation of these genes. Furthermore, extinction of gamma gene expression in adult life is not completely autonomous and is incomplete when HS2 is the only LCR element present.

Animals↗
PubMed2004

Spatiotemporal gene expression targeting with the TARGET and gene-switch systems in Drosophila.

Targeted gene expression has become a standard technique for the study of biological questions in Drosophila. Until recently, transgene expression could be targeted in the dimension of either time or space, but not both. Several new systems have recently been developed to direct transgene expression simultaneously in both time and space. We describe here two such systems that we developed in our laboratory. The first system provides a general method for temporal and regional gene expression targeting (TARGET) with the conventional GAL4-upstream activator sequence (UAS) system and a temperature-sensitive GAL80 molecule, which represses GAL4 transcriptional activity at permissive temperatures. The second system, termed Gene-Switch, is based on a GAL4-progesterone receptor chimera that is hormone-inducible. We have used both systems for simultaneous spatial and temporal rescue of memory dysfunction in the rutabaga (rut) memory mutant of Drosophila. In this protocol, we provide guidelines for the use of these two novel systems, which should have general utility in studying Drosophila biology and in using the fly as a model for human disease.

5' Flanking Region↗
PubMed2000

Identification of ligands and coligands for the ecdysone-regulated gene switch.

The ecdysone-inducible gene switch is a useful tool for modulating gene expression in mammalian cells and transgenic animals. We have identified inducers derived from plants as well as certain classes of insecticides that increase the versatility of this gene regulation system. Phytoecdysteroids share the favorable kinetics of steroids, but are inert in mammals. The gene regulation properties of one of these ecdysteroids have been examined in cell culture and in newly developed strains of ecdysone-system transgenic mice. Ponasterone A is a potent regulator of gene expression in cells and transgenic animals, enabling reporter genes to be turned on and off rapidly. A number of nonsteroidal insecticides have been identified that also activate the ecdysone system. Because the gene-controlling properties of the ecdysone switch are based on a heterodimer composed of a modified ecdysone receptor (VgEcR) and the retinoid X receptor (RXR), we have tested the effect of RXR ligands on the VgEcR/RXR complex. Used alone, RXR ligands display no activity on the ecdysone switch. However, when used in combination with a VgEcR ligand, RXR ligands dramatically enhance the absolute levels of induction. This property of the heterodimer has allowed the development of superinducer combinations that increase the dynamic range of the system.

Animals↗
PubMedDate not supplied

A human putative lymphocyte G0/G1 switch gene homologous to a rodent gene encoding a zinc-binding potential transcription factor.

G0S24 is a member of a set of genes (putative G0/G1 switch regulatory genes) that are expressed transiently within 1-2 hr of the addition of lectin or cycloheximide to human blood mononuclear cells. Comparison of a full-length cDNA sequence with the corresponding genomic sequence reveals an open reading frame of 326 amino acids, distributed across two exons. Potential phosphorylation sites include the sequence PSPTSPT, which resembles an RNA polymerase II repeat reported to be a target of the cell cycle control kinase cdc2. Comparison of the derived protein sequence with those of rodent homologs allows classification into three groups. Group 1 contains G0S24 and the rat and mouse TIS11 genes (also known as TTP, Nup475, and Zfp36). Members of this group have three tetraproline repeats. Groups 1 and 2 have a serine-rich region and an "arginine element" (RRLPIF) at the carboxyl terminus. All groups contain cysteine- and histidine-rich putative zinc finger domains and a serine-phenylalanine "SFS" domain similar to part of the large subunit of eukaryotic RNA polymerase II. Comparison of group 1 human and mouse genomic sequences shows high conservation in the 5' flank and exons. A CpG island suggests expression in the germ line. G0S24 has potential sites for transcription factors in the 5' flank and intron; these include a serum response element. Protein and genomic sequences show similarities with those of a variety of proteins involved in transcription, suggesting that the G0S24 product has a similar role.

Amino Acid Sequence↗
PubMed2004

Gene switching and the stability of odorant receptor gene choice.

Individual olfactory sensory neurons express only a single odorant receptor from a large family of genes, and this singularity is an essential feature in models of olfactory perception. We have devised a genetic strategy to examine the stability of receptor choice. We observe that immature olfactory sensory neurons that express a given odorant receptor can switch receptor expression, albeit at low frequency. Neurons that express a mutant receptor gene switch receptor transcription with significantly greater probability, suggesting that the expression of a functional odorant receptor elicits a feedback signal that terminates switching. This process of receptor gene switching assures that a neuron will ultimately express a functional receptor and that the choice of this receptor will remain stable for the life of the cell.

Animals↗
PubMed2005

Novel gene switches for targeted and timed expression of proteins of interest.

This article reports on the construction and analysis in vitro and in vivo of novel gene switches that can be used to achieve spatial as well as temporal control over the expression of a transgene of interest. The switches are expected to be functional in virtually any tissue and cell type. They consist of (a) a foreign or modified transactivator expressed under the dual control of a promoter or promoter cassette that is responsive to heat and the transactivator and (b) a promoter responsive to the transactivator for controlling the transgene of interest. A preferred gene switch of this type incorporated a mifepristone-dependent transactivator. This gene switch could be activated by a transient heat treatment in the presence of mifepristone. Activity increased with the intensity of the activating heat treatment and was found to persist for more than 6 days. The gene switch was essentially inactive prior to an activating heat treatment, in the absence or presence of mifepristone. Activated gene switch could be silenced by removal/withdrawal of mifepristone.

Gene Expression Regulation↗