scholarly journals A toolkit to generate inducible and interconvertible Drosophila transgenes

2020 ◽  
Author(s):  
Franz Wendler ◽  
Sangbin Park ◽  
Claire Hill ◽  
Alessia Galasso ◽  
Kathleen R. Chang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Binary Systems ◽  
Expression System ◽  
Fruit Fly ◽  
Regulatory Elements ◽  
Conditional Gene Expression ◽  
Dna Elements ◽  
Independent Site ◽  
Inducible Gene

ABSTRACTThe existence of three independent binary systems for conditional gene expression (Gal4/UAS; LexA/LexAop; QF/QUAST) has greatly expanded versatile genetic analyses in the fruit fly Drosophila melanogaster; however, the experimental application of these tools is limited by the need to generate multiple collections of non-interchangeable transgenic fly strains for each inducible gene expression system. To address this practical limitation, we developed a multipurpose modular vector that contains the regulatory elements from all three binary systems, enabling Gal4-, LexA- or QF-dependent expression of transgenes. Our methods also incorporate DNA elements that facilitate independent site-specific recombination and elimination of regulatory UAS, LexAop or QUAST modules with spatial and temporal control, thus offering unprecedented possibilities and logistical advantages for in vivo genetic modulation and efficient interconversion of transgenic fly lines.

scholarly journals First report of a tetracycline-inducible gene expression system for mollicutes

Microbiology ◽  
2010 ◽  
Vol 156 (1) ◽  
pp. 198-205 ◽  
Author(s):  
Marc Breton ◽  
Evelyne Sagné ◽  
Sybille Duret ◽  
Laure Béven ◽  
Christine Citti ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression System ◽  
Inducible Promoter ◽  
High Level Expression ◽  
Spiroplasma Citri ◽  
High Level ◽  
Tetr Repressor ◽  
Promoter System ◽  
Inducible Gene

Inducible promoter systems are powerful tools for studying gene function in prokaryotes but have never been shown to function in mollicutes. In this study we evaluated the efficacy of the tetracycline-inducible promoter Pxyl/tetO2 from Bacillus subtilis in controlling gene expression in two mollicutes, the plant pathogen Spiroplasma citri and the animal pathogen Mycoplasma agalactiae. An S. citri plasmid carrying the spiralin gene under the control of the xyl/tetO2 tetracycline-inducible promoter and the TetR repressor gene under the control of a constitutive spiroplasmal promoter was introduced into the spiralin-less S. citri mutant GII3-9a3. In the absence of tetracycline, expression of TetR almost completely abolished expression of spiralin from the xyl/tetO2 promoter. Adding tetracycline (>50 ng ml−1) to the medium induced high-level expression of spiralin. Interestingly, inducible expression of spiralin was also detected in vivo: in S. citri-infected leafhoppers fed on tetracycline-containing medium and in S. citri-infected plants watered with tetracycline. A similar construct was introduced into the M. agalactiae chromosome through transposition. Tetracycline-induced expression of spiralin proved the TetR-Pxyl/tetO2 system to be functional in the ruminant pathogen, suggesting that this tetracycline-inducible promoter system might be of general use in mollicutes.

scholarly journals Development of a Sensitive Gene Expression Reporter System and an Inducible Promoter-Repressor System for Clostridium acetobutylicum

2003 ◽  
Vol 69 (8) ◽  
pp. 4985-4988 ◽  
Author(s):  
Laurence Girbal ◽  
Isabelle Mortier-Barrière ◽  
Frédéric Raynaud ◽  
Céline Rouanet ◽  
Christian Croux ◽  
...  
Keyword(s):  
Gene Expression ◽  
Clostridium Acetobutylicum ◽  
Time Course ◽  
Specific Activity ◽  
Expression System ◽  
Regulatory System ◽  
Inducible Promoter ◽  
Reporter System ◽  
Inducible Gene

ABSTRACT A sensitive gene expression reporter system was developed for Clostridium acetobutylicum ATCC 824 by using a customized gusA expression cassette. In discontinuous cultures, time course profiles of β-glucuronidase specific activity reflected adequately in vivo dynamic up- and down-regulation of acidogenesis- and/or solventogenesis-associated promoter expression in C. acetobutylicum. Furthermore, a new inducible gene expression system was developed in C. acetobutylicum, based on the Staphylococcus xylosus xylose operon promoter-repressor regulatory system.

scholarly journals The CD11b promoter directs high-level expression of reporter genes in macrophages in transgenic mice [published erratum appears in Blood 1995 Apr 1;85(7):1983]

Blood ◽  
1995 ◽  
Vol 85 (2) ◽  
pp. 319-329 ◽  
Author(s):  
S Dziennis ◽  
RA Van Etten ◽  
HL Pahl ◽  
DL Morris ◽  
TL Rothstein ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Transgene Expression ◽  
Heterologous Gene Expression ◽  
Myeloid Cells ◽  
Reporter Genes ◽  
Regulatory Elements ◽  
Specific Expression ◽  
High Level

Abstract CD11b is the alpha chain of the Mac-1 integrin and is preferentially expressed in myeloid cells (neutrophils, monocytes, and macrophages). We have previously shown that the CD11b promoter directs cell-type- specific expression in myeloid lines using transient transfection assays. To confirm that these promoter sequences contain the proper regulatory elements for correct myeloid expression of CD11b in vivo, we have used the -1.7-kb human CD11b promoter to direct reporter gene expression in transgenic mice. Stable founder lines were generated with two different reporter genes, a Thy 1.1 surface marker and the Escherichia coli lacZ (beta-galactosidase) gene. Analysis of founders generated with each reporter demonstrated that the CD11b promoter was capable of driving high levels of transgene expression in murine macrophages for the lifetime of the animals. Similar to the endogenous gene, transgene expression was preferentially found in mature monocytes, macrophages, and neutrophils and not in myeloid precursors. These experiments indicate that the -1.7 CD11b promoter contains the regulatory elements sufficient for high-level macrophage expression. This promoter should be useful for targeting heterologous gene expression to mature myeloid cells.

scholarly journals Comparative gene expression profiling of mouse ovaries upon stimulation with natural equine chorionic gonadotropin (N-eCG) and tethered recombinant-eCG (R-eCG)

2020 ◽  
Author(s):  
Kwan-Sik Min ◽  
Jong-Ju Park ◽  
So-Yun Lee ◽  
Munkhzaya Byambaragchaa ◽  
Myung-Hwa Kang
Keyword(s):  
Gene Expression ◽  
Chorionic Gonadotropin ◽  
Expression Profiles ◽  
Expression System ◽  
Laboratory Animals ◽  
Metabolic Clearance ◽  
Qrt Pcr ◽  
Significant Difference ◽  
Equine Chorionic Gonadotropin

Abstract Background: Equine chorionic gonadotropin (eCG) induces super-ovulation in laboratory animals. Notwithstanding its extensive usage, limited information is available regarding the differences between the in vivo effects of natural eCG (N-eCG) and recombinant eCG (R-eCG). This study aimed to investigate the gene expression profiles of mouse ovaries upon stimulation with N-eCG and R-eCG produced from CHO-suspension (CHO-S) cells. R-eCG gene was constructed and transfected into CHO-S cells and quantified. Subsequently, we determined the metabolic clearance rate (MCR) of N-eCG and R-eCG up to 24 h after intravenous administration through the mice tail vein and identified differentially expressed genes in both ovarian tissues, via quantitative real-time PCR (qRT-PCR) and immunohistochemistry (IHC).Results: R-eCG was markedly expressed initially after transfection and maintained until recovery on day 9. Glycan chains were substantially modified in R-eCG protein produced from CHO-S cells and eliminated through PNGase F treatment. The MCR was higher for R-eCG than for N-eCG, and no significant difference was observed after 60 min. Notwithstanding their low concentrations, R-eCG and N-eCG were detected in the blood at 24h post-injection. Microarray analysis of ovarian tissue revealed that 20 of 12,816 genes assessed therein were significantly up-regulated and 43 genes were down-regulated by >2-fold in the group that received R-eCG (63 [0.49%] differentially regulated genes in total). The microarray results were concurrent with and hence validated by those of RT-PCR, qRT-PCR, and IHC analyses.Conclusions: The present results indicate that R-eCG can be adequately produced through a cell-based expression system through post-translational modification of eCG and can induce ovulation in vivo. These results provide novel insights into the molecular mechanisms underlying the up- or down-regulation of specific ovarian genes and the production of R-eCG with enhanced biological activity in vivo.

scholarly journals Synthetic gene regulatory networks in the opportunistic human pathogen Streptococcus pneumoniae

2019 ◽  
Author(s):  
Robin A. Sorg ◽  
Clement Gallay ◽  
Jan-Willem Veening
Keyword(s):  
Gene Expression ◽  
Streptococcus Pneumoniae ◽  
Regulatory Networks ◽  
Expression Patterns ◽  
Combinatorial Libraries ◽  
Regulatory Elements ◽  
Expression Control ◽  
Gene Expression Control

AbstractStreptococcus pneumoniae can cause disease in various human tissues and organs, including the ear, the brain, the blood and the lung, and thus in highly diverse and dynamic environments. It is challenging to study how pneumococci control virulence factor expression, because cues of natural environments and the presence of an immune system are difficult to simulate in vitro. Here, we apply synthetic biology methods to reverse-engineer gene expression control in S. pneumoniae. A selection platform is described that allows for straightforward identification of transcriptional regulatory elements out of combinatorial libraries. We present TetR- and LacI-regulated promoters that show expression ranges of four orders of magnitude. Based on these promoters, regulatory networks of higher complexity are assembled, such as logic AND and IMPLY gates. Finally, we demonstrate single-copy genome-integrated toggle switches that give rise to bimodal population distributions. The tools described here can be used to mimic complex expression patterns, such as the ones found for pneumococcal virulence factors, paving the way for in vivo investigations of the importance of gene expression control on the pathogenicity of S. pneumoniae.

scholarly journals Direct neuronal reprogramming by temporal identity factors

2021 ◽  
Author(s):  
Camille Boudreau-Pinsonneault ◽  
Awais Javed ◽  
Michel Fries ◽  
Pierre Mattar ◽  
Michel Cayouette
Keyword(s):  
Gene Expression ◽  
Expression System ◽  
Lineage Tracing ◽  
Developmental Competence ◽  
Rapid Screening ◽  
Mouse Retina ◽  
Genetic Lineage ◽  
Differentiated Cells ◽  
Temporal Identity

Temporal identity factors are sufficient to reprogram developmental competence of neural progenitors, but whether they could also reprogram the identity of fully differentiated cells is unknown. To address this question, we designed a conditional gene expression system combined with genetic lineage tracing that allows rapid screening of potential reprogramming factors in the mouse retina. Using this assay, we report that co-expression of the early temporal identity transcription factor Ikzf1, together with Ikzf4, another Ikaros family member, is sufficient to directly convert adult Muller glial cells into neuron-like cells in vivo, without inducing a proliferative progenitor state. scRNA-seq analysis shows that the reprogrammed cells share some transcriptional signatures with both cone photoreceptors and bipolar cells. Furthermore, we show that co-expression of Ikzf1 and Ikzf4 can reprogram mouse embryonic fibroblasts to induced neurons by remodeling chromatin and promoting a neuronal gene expression program. This work uncovers general neuronal reprogramming properties for temporal identity factors in differentiated cells, opening new opportunities for cell therapy development.

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