scholarly journals Systematic identification of a panel of strong promoter regions from Listeria monocytogenes for fine-tuning gene expression

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Qianyu Ji ◽  
Junfei Ma ◽  
Shuying Wang ◽  
Qing Liu
Keyword(s):  
Listeria Monocytogenes ◽  
Fluorescent Protein ◽  
Fine Tuning ◽  
Egfp Expression ◽  
Promoter Regions ◽  
Strong Promoter ◽  
Yield Increase ◽  
Promoter Library ◽  
Green Fluorescent ◽  
Systematic Identification

Abstract Background Attenuated Listeria monocytogenes (Lm) has been widely used as a vaccine vector in the prevention and treatment of pathogen infection and tumor diseases. In addition, previous studies have proved that the attenuated Lm can protect zebrafish from Vibrio infections, indicating that the attenuated Lm has a good application prospect in the field of aquatic vaccines. However, the limitation mainly lies in the lack of a set of well-characterized natural promoters for the expression of target antigens in attenuated Lm. Results In our study, candidate strong promoters were identified through RNA-seq analysis, and characterized in Lm through enhanced green fluorescent protein (EGFP). Nine native promoters that showed stronger activities than that of the known strong promoter P36 under two tested temperatures (28 and 37 °C) were selected from the set, and P29 with the highest activity was 24-fold greater than P36. Furthermore, we demonstrated that P29 could initiate EGFP expression in ZF4 cells and zebrafish embryos. Conclusions This well-characterized promoter library can be used to fine-tune the expression of different proteins in Lm. The availability of a well-characterized promoter toolbox of Lm is essential for the analysis of yield increase for biotechnology applications.

scholarly journals A new transgene mouse model using an extravesicular EGFP tag to elucidate the in vivo function of extracellular vesicles

2021 ◽  
Author(s):  
Mikkel Oernfeldt Noegaard ◽  
Lasse Bach Steffensen ◽  
Didde Hansen ◽  
Ernst-Martin Fuchtbauer ◽  
Morten Buch Engelund ◽  
...  
Keyword(s):  
Mouse Model ◽  
Extracellular Vesicles ◽  
Fluorescent Protein ◽  
Urine Samples ◽  
Egfp Expression ◽  
Reporter Mice ◽  
Plasma And Urine Samples ◽  
Co Precipitation ◽  
Green Fluorescent

The in vivo function of cell-derived extracellular vesicles (EVs) is challenging to establish since cell-specific EVs are difficult to isolate. We therefore created an EV reporter using CD9 to display enhanced green fluorescent protein (EGFP) on the EV surface. CD9-EGFP expression in cells did not affect EV size and concentration, but allowed for co-precipitation of EV markers TSG101 and ALIX from cell-conditioned medium by anti-GFP immunoprecipitation. We created a transgenic mouse where CD9-EGFP was inserted in the inverse orientation and double-floxed, ensuring Cre recombinase-dependent EV reporter expression. We crossed the EV reporter mice with mice expressing Cre ubiquitously (CMV-Cre), in cardiomyocytes (AMHC-Cre) and kidney epithelium (Pax8-Cre), respectively. The mice showed tissue-specific EGFP expression, and plasma and urine samples were used to immunoprecipitate EVs. CD9-EGFP EVs was detected in plasma samples from CMV-Cre/CD9-EGFP and AMHC-Cre/CD9-EGFP mice, but not in PAX8-Cre/CD9-EGFP mice. On the other hand, CD9-EGFP EVs were detected in urine samples from CMV-Cre/CD9-EGFP and PAX8-Cre/CD9-EGFP mice, but not AMHC-Cre/CD9-EGFP, indicating that plasma EVs are not filtered to the urine. In conclusion, our EV reporter mouse model enables Cre-dependent EV labeling, providing a new approach to study cell-specific EVs in vivo and gain new insight into their physiological and pathophysiological function.

scholarly journals Enhanced Green Fluorescent Protein as Selectable Marker of Retroviral-Mediated Gene Transfer in Immature Hematopoietic Bone Marrow Cells

Blood ◽  
1997 ◽  
Vol 90 (9) ◽  
pp. 3304-3315 ◽  
Author(s):  
Marti F.A. Bierhuizen ◽  
Yvonne Westerman ◽  
Trudi P. Visser ◽  
Wati Dimjati ◽  
Albertus W. Wognum ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Green Fluorescent Protein ◽  
Gene Transfer ◽  
Fluorescent Protein ◽  
Bone Marrow Cells ◽  
Retroviral Vector ◽  
Egfp Expression ◽  
Green Fluorescent ◽  
Marrow Cells

Abstract The further improvement of gene transfer into hematopoietic stem cells and their direct progeny will be greatly facilitated by markers that allow rapid detection and efficient selection of successfully transduced cells. For this purpose, a retroviral vector was designed and tested encoding a recombinant version of the Aequorea victoria green fluorescent protein that is enhanced for high-level expression in mammalian cells (EGFP). Murine cell lines (NIH 3T3, Rat2) and bone marrow cells transduced with this retroviral vector demonstrated a stable green fluorescence signal readily detectable by flow cytometry. Functional analysis of the retrovirally transduced bone marrow cells showed EGFP expression in in vitro clonogenic progenitors (GM-CFU), day 13 colony-forming unit-spleen (CFU-S), and in peripheral blood cells and marrow repopulating cells of transplanted mice. In conjunction with fluorescence-activated cell sorting (FACS) techniques EGFP expression could be used as a marker to select for greater than 95% pure populations of transduced cells and to phenotypically define the transduced cells using antibodies directed against specific cell-surface antigens. Detrimental effects of EGFP expression were not observed: fluorescence intensity appeared to be stable and hematopoietic cell growth was not impaired. The data show the feasibility of using EGFP as a convenient and rapid reporter to monitor retroviral-mediated gene transfer and expression in hematopoietic cells, to select for the genetically modified cells, and to track these cells and their progeny both in vitro and in vivo.

Elucidation of the distal convoluted tubule transcriptome identifies new candidate genes involved in renal Mg2+ handling

2013 ◽  
Vol 305 (11) ◽  
pp. F1563-F1573 ◽  
Author(s):  
Jeroen H. F. de Baaij ◽  
Marian J. Groot Koerkamp ◽  
Marla Lavrijsen ◽  
Femke van Zeeland ◽  
Hans Meijer ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Candidate Genes ◽  
Transient Receptor Potential ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Distal Convoluted Tubule ◽  
Fine Tuning ◽  
Complex Object ◽  
New Genes ◽  
Green Fluorescent

The kidney plays a key role in the maintenance of Mg2+ homeostasis. Specifically, the distal convoluted tubule (DCT) is instrumental in the fine-tuning of renal Mg2+ handling. In recent years, hereditary Mg2+ transport disorders have helped to identify important players in DCT Mg2+ homeostasis. Nevertheless, several proteins involved in DCT-mediated Mg2+ reabsorption remain to be discovered, and a full expression profile of this complex nephron segment may facilitate the discovery of new Mg2+-related genes. Here, we report Mg2+-sensitive expression of the DCT transcriptome. To this end, transgenic mice expressing enhanced green fluorescent protein under a DCT-specific parvalbumin promoter were subjected to Mg2+-deficient or Mg2+-enriched diets. Subsequently, the Complex Object Parametric Analyzer and Sorter allowed, for the first time, isolation of enhanced green fluorescent protein-positive DCT cells. RNA extracts thereof were analyzed by DNA microarrays comparing high versus low Mg2+ to identify Mg2+ regulatory genes. Based on statistical significance and a fold change of at least 2, 46 genes showed differential expression. Several known magnesiotropic genes, such as transient receptor potential cation channel, subfamily M, member 6 ( Trpm6), and Parvalbumin, were upregulated under low dietary Mg2+. Moreover, new genes were identified that are potentially involved in renal Mg2+ handling. To confirm that the selected candidate genes were regulated by dietary Mg2+ availability, the expression levels of solute carrier family 41, member 3 ( Slc41a3), pterin-4 α-carbinolamine dehydratase/dimerization cofactor of hepatocyte nuclear factor-1α ( Pcbd1), TBC1 domain family, member 4 ( Tbc1d4), and uromodulin ( Umod) were determined by RT-PCR analysis. Indeed, all four genes show significant upregulation in the DCT of mice fed a Mg2+-deficient diet. By elucidating the Mg2+-sensitive DCT transcriptome, new candidate genes in renal Mg2+ handling have been identified.

Persistence of eGFP Marked Bone Marrow Cells in Long-Term Hematopoiesis.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2111-2111
Author(s):  
Ingo H. Pilz ◽  
Manfred Schmidt ◽  
Claudia Ball ◽  
Hanno Glimm ◽  
Fritz von Weizsäcker ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Alkylating Agent ◽  
Fluorescent Protein ◽  
Bone Marrow Cells ◽  
Egfp Expression ◽  
Hind Limbs ◽  
Long Term Follow Up ◽  
Green Fluorescent

Abstract To study transplanted unperturbed and mobilized long-term hematopoiesis after selection with an alkylating agent, bone marrow (BM) from 5 C57BL/6J mice was pooled, repeatedly transduced with retroviruses encoding the alkylating agent resistance protein O6-Methylguanine-DNA and enhanced green fluorescent protein (eGFP) as an easily traceable marker. Between 1 to 9x105 transfected BM cells were transplanted into 15 myeloablatively irradiated sex-mismatched C57BL/6J mice. Subsequently, 3 to 4 selection rounds with BCNU/O6-BG were carried out, enriching eGFP marked hematopoiesis in these mice up to 70–90%. Between 1 and 7x107BM cells of different mice were transplanted according to marrow location into groups of 5 sex-matched Bri44[1] mice. Two mice each received BM from the hind limbs, two from the pelvis and one received cells from the spleen, only, respectively. Altogether the study comprised 15 groups divided into 6 female and 9 male groups. Of these, 4 male and 3 female groups received 3 HSC-mobilization courses with G-CSF at intervals of 2 months starting 3 month after transplantation. Hematopoiesis in the other fraction remained unperturbed. During the observation period of 11–14 months in these tertiary recipients, repeated FACS analyses as well as linear amplification mediated (LAM) PCRs were carried out to track the clonal contributions. A decrease in the percentage of eGFP expressing marked hematopoiesis was observed in most cases. However, eGFP expression never disappeared altogether and could still be detected in the different hematopoietic lineages and successfully sorted for further analyses by MoFlo (Dako-Cytomation). Assessment of the clonal status of the Bri44 by LAM-PCR displayed interesting results. In some mice a decline in clone numbers was observed, whereas clone numbers remained stable in others. Tertiary transplantation with long-term follow-up indicates that this observation may be related to the transplantation of limited long-term repopulating clone numbers and progenitor cell exhaustion over time.

Validation of Apple Cider Pasteurization Treatments against Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes

2001 ◽  
Vol 64 (11) ◽  
pp. 1679-1689 ◽  
Author(s):  
PEGGY P. MAK ◽  
BARBARA H. INGHAM ◽  
STEVEN C. INGHAM
Keyword(s):  
Escherichia Coli ◽  
New York ◽  
Listeria Monocytogenes ◽  
Fluorescent Protein ◽  
Escherichia Coli O157 ◽  
Salmonella Spp ◽  
Apple Cider ◽  
E Coli ◽  
Log Reduction ◽  
Green Fluorescent

Time and temperature pasteurization conditions common in the Wisconsin cider industry were validated using a six-strain cocktail of Escherichia coli O157:H7 and acid-adapted E. coli O157:H7 in pH- and ∘Brix-adjusted apple cider. Strains employed were linked to outbreaks (ATCC 43894 and 43895, C7927, and USDA-FSIS-380–94) or strains engineered to contain the gene for green fluorescent protein (pGFP ATCC 43894 and pGFP ATCC 43889) for differential enumeration. Survival of Salmonella spp. (CDC 0778, CDC F2833, and CDC HO662) and Listeria monocytogenes (H0222, F8027, and F8369) was also evaluated. Inoculated cider of pH 3.3 or 4.1 and 11 or 14°Brix was heated under conditions ranging from 60°C for 14 s to 71.1°C for 14 s. A 5-log reduction of nonadapted and acid-adapted E. coli O157:H7 was obtained at 68.1°C for 14 s. Lower temperatures, or less time at 68.1°C, did not ensure a 5-log reduction in E. coli O157:H7. A 5-log reduction was obtained at 65.6°C for 14 s for Salmonella spp. L. monocytogenes survived 68.1°C for 14 s, but survivors died in cider within 24 h at 4°C. Laboratory results were validated with a surrogate E. coli using a bench-top plate heat-exchange pasteurizer. Results were further validated using fresh unpasteurized commercial ciders. Consumer acceptance of cider pasteurized at 68.1°C for 14 s (Wisconsin recommendations) and at 71.1°C for 6 s (New York recommendations) was not significantly different. Hence, we conclude that 68.1°C for 14 s is a validated treatment for ensuring adequate destruction of E. coli O157:H7, Salmonella spp., and L. monocytogenes in apple cider.

Efficient transduction of human hematopoietic repopulating cells generating stable engraftment of transgene-expressing cells in NOD/SCID mice

Blood ◽  
2000 ◽  
Vol 95 (10) ◽  
pp. 3085-3093 ◽  
Author(s):  
Jordi Barquinero ◽  
José Carlos Segovia ◽  
Manuel Ramı́rez ◽  
Ana Limón ◽  
Guillermo Güenechea ◽  
...  
Keyword(s):  
Southern Blotting ◽  
Fluorescent Protein ◽  
Leukemia Virus ◽  
Scid Mice ◽  
Egfp Expression ◽  
Facs Analysis ◽  
Insertion Sites ◽  
Cord Blood Cells ◽  
Gibbon Ape Leukemia Virus ◽  
Green Fluorescent

In an attempt to develop efficient procedures of human hematopoietic gene therapy, retrovirally transduced CD34+ cord blood cells were transplanted into NOD/SCID mice to evaluate the repopulating potential of transduced grafts. Samples were prestimulated on Retronectin-coated dishes and infected with gibbon ape leukemia virus (GALV)-pseudotyped FMEV vectors encoding the enhanced green fluorescent protein (EGFP). Periodic analyses of bone marrow (BM) from transplanted recipients revealed a sustained engraftment of human hematopoietic cells expressing the EGFP transgene. On average, 33.6% of human CD45+ cells expressed the transgene 90 to120 days after transplantation. Moreover, 11.9% of total NOD/SCID BM consisted of human CD45+ cells expressing the EGFP transgene at this time. The transplantation of purified EGFP+ cells increased the proportion of CD45+ cells positive for EGFP expression to 57.7% at 90 to 120 days after transplantation. At this time, 18.9% and 4.3% of NOD/SCID BM consisted of CD45+/EGFP+ and CD34+/EGFP+ cells, respectively. Interestingly, the transplantation of EGFP− cells purified at 24 hours after infection also generated a significant engraftment of CD45+/EGFP+ and CD34+/EGFP+ cells, suggesting that a number of transduced repopulating cells did not express the transgene at that time. Molecular analysis of NOD/SCID BM confirmed the high levels of engraftment of human transduced cells deduced from FACS analysis. Finally, the analysis of the provirus insertion sites by conventional Southern blotting indicated that the human hematopoiesis in the NOD/SCID BM was predominantly oligoclonal.

Intestine-specific expression of green fluorescent protein-tagged cathepsin B: proof-of-principle experiments

2008 ◽  
Vol 389 (8) ◽  
Author(s):  
Kristina Mayer ◽  
Maria E. Iolyeva ◽  
Ulf Meyer-Grahle ◽  
Klaudia Brix
Keyword(s):  
Transcription Factor ◽  
Green Fluorescent Protein ◽  
Cathepsin B ◽  
Fluorescent Protein ◽  
Regulatory Elements ◽  
Egfp Expression ◽  
Reporter Protein ◽  
Specific Expression ◽  
Green Fluorescent ◽  
Proof Of Principle

Abstract We hypothesized that tissue-specific expression of cathepsin B-enhanced green fluorescent protein (CB-EGFP) can be driven by the A33-antigen promoter that contains positive cis-regulatory elements, including caudal-related homeobox (CDX) binding sites. The intestine-specific transcription factor Cdx1 is crucial for A33-antigen promoter activation and could thereby induce expression of CB-EGFP. This concept was tested by construction of the vector pA33-CathB-EGFP encoding CB-EGFP downstream of the A33-antigen promoter. Its Cdx1 dependence, as an indication of its intestine-specific expression, was tested in Cdx1-negative CHO-K1 cells. Cdx1 expression was achieved upon transfection with pCdx1-DsRed-Express and was indicated by red fluorescence of the simultaneously translated reporter protein. Immunolabeling with Cdx1-specific antibodies showed correct targeting of the transcription factor to its point of action in nuclei of transfected cells. Co-transfection experiments with plasmids pA33-CathB-EGFP and pCdx1-DsRed-Express confirmed the hypothesis that Cdx1 indeed activates CB-EGFP expression in a manner dependent on the A33-antigen promoter. Co-localization with compartment-specific markers and subcellular fractionation confirmed CB-EGFP trafficking along the expected route to endolysosomal compartments. Hence, the A33-antigen promoter represents a potent tool for induction of Cdx1-dependent CB-EGFP expression in vitro. Our proof-of-principle studies confirm the suitability of this approach in visualizing protease transport in Cdx1-positive tissues of the gastrointestinal tract.

scholarly journals Enhancement of β-Globin Locus Control Region-Mediated Transactivation by Mitogen-Activated Protein Kinases through Stochastic and Graded Mechanisms

1999 ◽  
Vol 19 (8) ◽  
pp. 5565-5575 ◽  
Author(s):  
E. Camilla Forsberg ◽  
Tatiana N. Zaboikina ◽  
Wayne K. Versaw ◽  
Natalie G. Ahn ◽  
Emery H. Bresnick
Keyword(s):  
Flow Cytometry ◽  
Control Region ◽  
Fluorescent Protein ◽  
Mapk Pathway ◽  
Locus Control Region ◽  
Mitogen Activated Protein Kinase ◽  
Egfp Expression ◽  
Enhancer Activity ◽  
Mitogen Activated Protein ◽  
Green Fluorescent

ABSTRACT Activation of the mitogen-activated protein kinase (MAPK) pathway enhances long-range transactivation by the β-globin locus control region (LCR) (W. K. Versaw, V. Blank, N. M. Andrews, and E. H. Bresnick, Proc. Natl. Acad. Sci. USA 95:8756–8760, 1998). The enhancement requires tandem recognition sites for the hematopoietic transcription factor NF-E2 within the hypersensitive site 2 (HS2) subregion of the LCR. To distinguish between mechanisms of induction involving the activation of silent promoters or the increased efficacy of active promoters, we analyzed basal and MAPK-stimulated HS2 enhancer activity in single, living cells. K562 erythroleukemia cells stably transfected with constructs containing the human Aγ-globin promoter linked to an enhanced green fluorescent protein (EGFP) reporter, with or without HS2, were analyzed for EGFP expression by flow cytometry. When most cells in a population expressed EGFP, MAPK augmented the activity of active promoters. However, under conditions of silencing, in which cells reverted to a state with no measurable EGFP expression, MAPK activated silent promoters. Furthermore, studies of populations of EGFP-expressing and non-EGFP-expressing cells isolated by flow cytometry showed that MAPK activation converted nonexpressing cells into expressing cells and increased expression in expressing cells. These results support a model in which MAPK elicits both graded and stochastic responses to increase HS2-mediated transactivation from single chromatin templates.

scholarly journals Mycobacterium avium Genes Associated with the Ability To Form a Biofilm

2006 ◽  
Vol 72 (1) ◽  
pp. 819-825 ◽  
Author(s):  
Yoshitaka Yamazaki ◽  
Lia Danelishvili ◽  
Martin Wu ◽  
Molly MacNab ◽  
Luiz E. Bermudez
Keyword(s):  
Mycobacterium Avium ◽  
Fatty Acid Biosynthesis ◽  
Fluorescent Protein ◽  
Tca Cycle ◽  
Promoter Library ◽  
The Tca Cycle ◽  
Important Pathway ◽  
Mutant Bank ◽  
Green Fluorescent ◽  
Mycobacterial Strain

ABSTRACT Mycobacterium avium is widely distributed in the environment, and it is chiefly found in water and soil. M. avium, as well as Mycobacterium smegmatis, has been recognized to produce a biofilm or biofilm-like structure. We screened an M. avium green fluorescent protein (GFP) promoter library in M. smegmatis for genes involved in biofilm formation on polyvinyl chloride (PVC) plates. Clones associated with increased GFP expression ≥2.0-fold over the baseline were sequenced. Seventeen genes, most encoding proteins of the tricarboxylic acid (TCA) cycle and GDP-mannose and fatty acid biosynthesis, were identified. Their regulation in M. avium was confirmed by examining the expression of a set of genes by real-time PCR after incubation on PVC plates. In addition, screening of 2,000 clones of a transposon mutant bank constructed using M. avium strain A5, a mycobacterial strain with the ability to produce large amounts of biofilm, revealed four mutants with an impaired ability to form biofilm. Genes interrupted by transposons were homologues of M. tuberculosis 6-oxodehydrogenase (sucA), enzymes of the TCA cycle, protein synthetase (pstB), enzymes of glycopeptidolipid (GPL) synthesis, and Rv1565c (a hypothetical membrane protein). In conclusion, it appears that GPL biosynthesis, including the GDP-mannose biosynthesis pathway, is the most important pathway involved in the production of M. avium biofilm.

Sign in / Sign up

Export Citation Format

Share Document