Temporal Control Of Expression Research Articles

Generation of a Tetracycline Regulated Mouse Model of MYC-Induced T-Cell Acute Lymphoblastic Leukemia.

The tetracycline regulatory system provides a tractable strategy to interrogate the role of oncogenes in the initiation, maintenance, and regression of tumors through both spatial and temporal control of expression. This approach has several potential advantages over conventional methods to generate genetically engineered mouse models. First, continuous constitutive overexpression of an oncogene can be lethal to the host impeding further study. Second, constitutive overexpression fails to model adult onset of disease. Third, constitutive deletion does not permit, whereas conditional overexpression of an oncogene enables the study of the consequences of restoring expression of an oncogene back to endogenous levels. Fourth, the conditional activation of oncogenes enables examination of specific and/or developmental state-specific consequences.Hence, by allowing precise control of when and where a gene is expressed, the tetracycline regulatory system provides an ideal approach for the study of putative oncogenes in the initiation as well as the maintenance of tumorigenesis and the examination of the mechanisms of oncogene addiction. In this protocol, we describe the methods involved in the development of a conditional mouse model of MYC-induced T-cell acute lymphoblastic leukemia.

Intein-mediated cytoplasmic reconstitution of a split toxin enables selective cell ablation in mixed populations and tumor xenografts

The application of proteinaceous toxins for cell ablation is limited by their high on- and off-target toxicity, severe side effects, and a narrow therapeutic window. The selectivity of targeting can be improved by intein-based toxin reconstitution from two dysfunctional fragments provided their cytoplasmic delivery via independent, selective pathways. While the reconstitution of proteins from genetically encoded elements has been explored, exploiting cell-surface receptors for boosting selectivity has not been attained. We designed a robust splitting algorithm and achieved reliable cytoplasmic reconstitution of functional diphtheria toxin from engineered intein-flanked fragments upon receptor-mediated delivery of one of them to the cells expressing the counterpart. Retargeting the delivery machinery toward different receptors overexpressed in cancer cells enables selective ablation of specific subpopulations in mixed cell cultures. In a mouse model, the transmembrane delivery of a split-toxin construct potently inhibits the growth of xenograft tumors expressing the split counterpart. Receptor-mediated delivery of engineered split proteins provides a platform for precise therapeutic and experimental ablation of tumors or desired cell populations while also greatly expanding the applicability of the intein-based protein transsplicing.

Gene Therapy Tools for Brain Diseases.

Neurological disorders affecting the central nervous system (CNS) are still incompletely understood. Many of these disorders lack a cure and are seeking more specific and effective treatments. In fact, in spite of advancements in knowledge of the CNS function, the treatment of neurological disorders with modern medical and surgical approaches remains difficult for many reasons, such as the complexity of the CNS, the limited regenerative capacity of the tissue, and the difficulty in conveying conventional drugs to the organ due to the blood–brain barrier. Gene therapy, allowing the delivery of genetic materials that encodes potential therapeutic molecules, represents an attractive option. Gene therapy can result in a stable or inducible expression of transgene(s), and can allow a nearly specific expression in target cells. In this review, we will discuss the most commonly used tools for the delivery of genetic material in the CNS, including viral and non-viral vectors; their main applications; their advantages and disadvantages. We will discuss mechanisms of genetic regulation through cell-specific and inducible promoters, which allow to express gene products only in specific cells and to control their transcriptional activation. In addition, we will describe the applications to CNS diseases of post-transcriptional regulation systems (RNA interference); of systems allowing spatial or temporal control of expression [optogenetics and Designer Receptors Exclusively Activated by Designer Drugs (DREADDs)]; and of gene editing technologies (CRISPR/Cas9, Zinc finger proteins). Particular attention will be reserved to viral vectors derived from herpes simplex type 1, a potential tool for the delivery and expression of multiple transgene cassettes simultaneously.

Comparison of promoters in transgenic rice

Reports of the use of rice storage protein gene promoters to express transgenes in rice grain have demonstrated that rice grain can be used as a production platform for end-use quality or seed-based edible vaccines. The generation of transgenic rice with multitraits (gene stacking), which requires the use of multiple transgenes under the control of different promoters, necessitates the use of promoters from rice and other cereals, as this is highly advantageous in reducing homology-based transcriptional gene silencing. Using the green fluorescent protein gene (gfp) as a reporter gene and a transgenic rice platform, promoters of storage protein and non-storage protein genes from barley, wheat and rice were compared with regard to their spatial and temporal control of expression. Storage protein promoters from barley (549-bp B-hordein and 433-bp D-hordein) and wheat (425-bp high-molecular-weight glutenin) directed the expression of green fluorescent protein (GFP) in endosperm but not embryo; however, expression was leaky, as it was also observed in seed maternal tissues, leaf and root tissues. As expected, the rice promoters (1350-bp alpha-glutelin B-1 and 1007-bp alpha-globulin) directed the endosperm-specific expression of GFP in transgenic rice. Our results indicate that seed-specific promoters from barley and wheat, although containing endosperm and GCN4 motifs, which are important for endosperm-specific expression in rice, may not be spatially regulated in the same manner as they are in their native species. The analysis of GFP expression under the control of various promoters in rice grain indicates that promoters from other cereals can drive high levels of endosperm-specific expression in rice, but their utility for seed-specific expression may depend on their tissue specificity.

Genetic Labeling: New Approaches to Creating a Gonadotroph “ID”

Genetic Labeling: New Approaches to Creating a Gonadotroph “ID”

Transcriptional Modulation of Genes Encoding Structural Characteristics of Differentiating Enterocytes During Development of a Polarized Epithelium In Vitro

Although there is considerable evidence implicating posttranslational mechanisms in the development of epithelial cell polarity, little is known about the patterns of gene expression and transcriptional regulation during this process. We characterized the temporal program of gene expression during cell-cell adhesion-initiated polarization of human Caco-2 cells in tissue culture, which develop structural and functional polarity similar to that of enterocytes in vivo. A distinctive switch in gene expression patterns occurred upon formation of cell-cell contacts between neighboring cells. Expression of genes involved in cell proliferation was down-regulated concomitant with induction of genes necessary for functional specialization of polarized epithelial cells. Transcriptional up-regulation of these latter genes correlated with formation of important structural and functional features in enterocyte differentiation and establishment of structural and functional cell polarity; components of the apical microvilli were induced as the brush border formed during polarization; as barrier function was established, expression of tight junction transmembrane proteins peaked; transcripts encoding components of the apical, but not the basal-lateral trafficking machinery were increased during polarization. Coordinated expression of genes encoding components of functional cell structures were often observed indicating temporal control of expression and assembly of multiprotein complexes.

Conditional and inducible transgene expression in mice through the combinatorial use of Cre-mediated recombination and tetracycline induction

Here we describe a triple transgenic mouse system, which combines the tissue specificity of any Cre-transgenic line with the inducibility of the reverse tetracycline transactivator (rtTA)/tetracycline-responsive element (tet-O)-driven transgenes. To ensure reliable rtTA expression in a broad range of cell types, we have targeted the rtTA transgene into the ROSA26 locus. The rtTA expression, however, is conditional to a Cre recombinase-mediated excision of a STOP region from the ROSA26 locus. We demonstrate the utility of this technology through the inducible expression of the vascular endothelial growth factor (VEGF-A) during embryonic development and postnatally in adult mice. Our results of adult induction recapitulate several different hepatic and immune cell pathological phenotypes associated with increased systemic VEGF-A protein levels. This system will be useful for studying genes in which temporal control of expression is necessary for the discovery of the full spectrum of functions. The presented approach abrogates the need to generate tissue-specific rtTA transgenes for tissues where well-characterized Cre lines already exist.

Expression of the chemically inducible maize GST-27 promoter in potato

Chemically inducible gene regulation systems provide a mechanism for a temporal control of expression of transgenes. In this study expression from the herbicide safener inducible maize GST-27 promoter was tested in potato, with the aim of using this to provide inducible expression of transgenes implicated in dormancy control. A binary vector comprising 3.8 kb of the GST-27 promoter was fused to the β-glucuronidase (GUS) reporter gene and transformed into potato. Application of the chemical inducer elevated the expression of GUS up to 40 fold in leaf tissue. However in stems, roots and tubers the GST-27 promoter caused high levels of expression of GUS in the absence of safener, demonstrating that in these tissues it acts as a constitutive promoter. A deleted promoter region of the GST-27 promoter displayed a similar expression pattern. Analysis of GUS activity in dormant and sprouting tubers showed that the GST-27 promoter was a strong constitutive promoter throughout the tuber life cycle.

Regulation of the Caulobacter crescentus dnaKJ operon.

The bacterial heat shock proteins DnaK and DnaJ are members of a class of molecular chaperones that are required for a wide variety of cellular functions at normal growth temperatures. In Caulobacter crescentus, the expression of the dnaKJ operon is regulated both temporally during the normal cell cycle and by heat shock. Analysis of deletions and base substitutions in the 5' region of the operon established the presence of two functional promoters: a heat shock-inducible promoter, P1, with characteristics of a sigma 32 promoter, and an adjacent sigma 70-like promoter, P2. Transcription initiating at the sigma 70-like promoter is under strict temporal control, whereas transcription initiating at the heat shock promoter at 30 degrees C is not. Transcription of dnaKJ occurs during a short period in the cell cycle, concomitant with the onset of DNA replication. Deletions in the 5' region have also revealed that all cis-acting sites required for temporal control of transcription reside within 50 bases of the P2 start site. Transcripts initiating from either the P1 or the P2 promoter have an RNA leader sequence with a high probability of forming an extensive secondary structure. Deletion of this leader sequence resulted in an increased rate of expression in both transcriptional and translational fusions. Although the temporal control of expression at physiological temperatures is not affected by the presence or absence of the leader sequence, changes in mRNA secondary structure may contribute to the modulation of DnaK and DnaJ levels at normal temperatures and during heat shock.