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Search results for: Cre Expression Plasmid 705-Cre;cm

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#33334884   2020/12/17 To Up

Genome editing demonstrates that the -5 kb Nanog enhancer regulates Nanog expression by modulating RNAPII initiation and/or recruitment.

Transcriptional enhancers have been defined by their ability to operate independent of distance and orientation in plasmid-based reporter assays of gene expression. Currently, histone marks are used to identify and define enhancers but do not consider the endogenous role of an enhancer in the context of native chromatin. We employed a combination of genomic editing, single cell analyses, and sequencing approaches to investigate a -associated -regulatory element (CRE) which has been reported by others to be either an alternative promoter or a super-enhancer (SE). We first demonstrate both distance and orientation independence in native chromatin, eliminating the issues raised with plasmid-based approaches. We next demonstrate that the dominant SE modulates globally and operates by recruiting and/or initiating RNA Polymerase II. Our studies have important implications to how transcriptional enhancers are defined and how they regulate gene expression.
Puja Agrawal, Steven Blinka, Kirthi Pulakanti, Michael H Reimer, Cary Stelloh, Alison Meyer, Sridhar Rao

2201 related Products with: Genome editing demonstrates that the -5 kb Nanog enhancer regulates Nanog expression by modulating RNAPII initiation and/or recruitment.

50ul100ul250ul500IU50ul (1mg/ml)100 μg50ul (1mg/ml)

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#33248294   2020/11/25 To Up

Adaptive thermogenesis in brown adipose tissue involves activation of pannexin-1 channels.

Brown adipose tissue (BAT) is specialized in thermogenesis. The conversion of energy into heat in brown adipocytes proceeds via stimulation of β-adrenergic receptor (βAR)-dependent signaling and activation of mitochondrial uncoupling protein 1 (UCP1). We have previously demonstrated a functional role for pannexin-1 (Panx1) channels in white adipose tissue; however, it is not known whether Panx1 channels play a role in the regulation of brown adipocyte function. Here, we tested the hypothesis that Panx1 channels are involved in brown adipocyte activation and thermogenesis.
Subramanian Senthivinayagam, Vlad Serbulea, Clint M Upchurch, Renata Polanowska-Grabowska, Suresh K Mendu, Srabani Sahu, Prathiba Jayaguru, Kevin W Aylor, Mahendra D Chordia, Limor Steinberg, Nathaniel Oberholtzer, Seichii Uchiyama, Noriko Inada, Ulrike M Lorenz, Thurl E Harris, Susanna R Keller, Akshaya K Meher, Alexandra Kadl, Bimal N Desai, Bijoy K Kundu, Norbert Leitinger

1334 related Products with: Adaptive thermogenesis in brown adipose tissue involves activation of pannexin-1 channels.

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#33229326   // To Up

[Construction of a striatum-specific Slc20a2 gene knockout mice model by CRISPR/Cas9 AAV system].

Primary familial brain calcification (PFBC) is a chronic progressive neurogenetic disorder. Its clinical symptoms mainly include dyskinesia, cognitive disorder and mental impairment; and the pathogenesis remains unclear. Studies have shown that SLC20A2 is the most common pathogenic gene of the disease. Since the Slc20a2 gene knockout mouse model could result in fetal growth restriction, in order to better understand the pathogenesis of PFBC, the present study used the CRISPR/Cas9 technology to construct a conditional knockout model of Slc20a2 gene in the striatum of mice. First, three sgRNAs (single guide RNAs) were designed to target the exon3 of Slc20a2 gene. The activity of the respective sgRNA was verified by constructing expression plasmids, transfecting cells and Surveyor assay. Second, the SgRNA with the highest activity was selected to generate the recombinant AAV-Cre virus, which was injected into the striatum of mice by stereotactic method. In vitro experiments showed that the three sgRNAs could effectively mediate Cas9 cleavage of the respective target DNA. The activity of Cre recombinase of the AAV-Cre was confirmed by immunofluorescence assay. Immunohistochemistry, TA clone, high-throughput sequencing and Western blot were used to detect and evaluate the efficiency of Slc20a2 gene knockout. The results showed that the Slc20a2 expression in the striatum of mice in the experimental group decreased significantly. In this study, three sgRNAs capable of knockout of Slc20a2 were successfully designed, and the conditional knockout of the Slc20a2 gene in the striatum of mouse was successfully established by the CRISPR/Cas9 technology, thereby providing an effective animal model for studying the pathogenesis of PFBC.
Min Ting Lin, Lu Lu Lai, Miao Zhao, Bi Wei Lin, Xiang Ping Yao

2148 related Products with: [Construction of a striatum-specific Slc20a2 gene knockout mice model by CRISPR/Cas9 AAV system].

50 ug2 Pieces/Box 25UG50 ug100ug Lyophilized50 ug4 Membranes/Box0.1 mg 6 ml Ready-to-use 50 ug2 Pieces/Box

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#33215257   2020/11/20 To Up

Catabolite responsive elements as a strategy for the control of heterologous gene expression in lactobacilli.

Genes involved in the transport and catabolism of carbohydrates are usually controlled through the binding of the catabolite control protein A (CcpA) to the catabolite-responsive elements (cre) of target genes in Gram-positive bacteria. In this work, we show how the elimination of the cre sites in Lactobacillus casei BL23 promoters induced by sorbitol (PgutF), maltose (PmalL), and myo-inositol (PiolT) allowed the induction of gene expression in media supplemented with sorbitol, maltose, and myo-inositol, respectively, even in the presence of glucose. This was studied using plasmids encoding the anaerobic fluorescent protein evoglow-Pp1 as a reporter. In addition, gutF cre site was introduced into a bile inducible promoter (P16090) and into the constitutive promoter of the elongation factor P (PEf-P) of L. casei BL23. The existence of the cre site blocked gene expression in the P16090 inducible promoter in the presence of glucose, while it had no influence on the expression of the PEf-P constitutive one. These results demonstrated that the introduction or elimination of cre sites in inducible promoters allows the control and modification of their heterologous genetic expression, showing how the cre site, the transcriptional regulator, and CcpA interact to control gene expression in inducible genes. KEY POINTS: • Cre sequences regulate gene expression in inducible promoters in L. casei BL23. • Cre sites do not affect gene expression in constitutive promoters in L. casei BL23. • Cre sequences could control heterologous genic expression in lactobacilli.
Susana Langa, Ángela Peirotén, Juan Luis Arqués, José María Landete

1588 related Products with: Catabolite responsive elements as a strategy for the control of heterologous gene expression in lactobacilli.

300 units100 tests96 samples100 tests10 Plates

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#33168749   2020/11/09 To Up

Discovery of small-molecule inhibitors of multidrug-resistance plasmid maintenance using a high-throughput screening approach.

Carbapenem-resistant Enterobacteriaceae (CRE) are multidrug-resistant pathogens for which new treatments are desperately needed. Carbapenemases and other types of antibiotic resistance genes are carried almost exclusively on large, low-copy-number plasmids (pCRE). Accordingly, small molecules that efficiently evict pCRE plasmids should restore much-needed treatment options. We therefore designed a high-throughput screen to identify such compounds. A synthetic plasmid was constructed containing the plasmid replication machinery from a representative CRE isolate as well as a fluorescent reporter gene to easily monitor plasmid maintenance. The synthetic plasmid was then introduced into an K12 host. We used this screening strain to test a library of over 12,000 known bioactive agents for molecules that selectively reduce plasmid levels relative to effects on bacterial growth. From 366 screen hits we further validated the antiplasmid activity of kasugamycin, an aminoglycoside; CGS 15943, a nucleoside analog; and Ro 90-7501, a bibenzimidazole. All three compounds exhibited significant antiplasmid activity including up to complete suppression of plasmid replication and/or plasmid eviction in multiple orthogonal readouts and potentiated activity of the carbapenem, meropenem, against a strain carrying the large, pCRE plasmid from which we constructed the synthetic screening plasmid. Additionally, we found kasugamycin and CGS 15943 blocked plasmid replication, respectively, by inhibiting expression or function of the plasmid replication initiation protein, RepE. In summary, we validated our approach to identify compounds that alter plasmid maintenance, confer resensitization to antimicrobials, and have specific mechanisms of action.
Katelyn E Zulauf, James E Kirby

2804 related Products with: Discovery of small-molecule inhibitors of multidrug-resistance plasmid maintenance using a high-throughput screening approach.

400Tests2 Sample Kit2 Sample Kit8 Sample Kit4 Sample KitUp to 200 ml cultures4 Sample Kit

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#33166564   2020/11/06 To Up

Cre/LoxP-HBV plasmids generating recombinant covalently closed circular DNA genome upon transfection.

New therapies against hepatitis B virus (HBV) require the elimination of covalently closed circular DNA (cccDNA), the episomal HBV genome. HBV plasmids containing an overlength 1.3-mer genome and bacterial backbone (pHBV1.3) are used in many different models, but do not replicate the unique features of cccDNA. Since the stable cccDNA pool is a barrier to HBV eradication in patients, we developed a recombinant circular HBV genome (rcccDNA) to mimic the cccDNA using Cre/LoxP technology. We validated four LoxP insertion sites into the HBV genome using hydrodynamic tail vein injection into murine liver, demonstrating high levels of HBV surface antigen (HBsAg) and HBV DNA expression with rcccDNA formation. HBsAg expression from rcccDNA was >30,000 ng/mL over 78 days, while HBsAg-expression from pHBV1.3 plasmid DNA declined from 2753 ng/mL to 131 ng/mL over that time in immunodeficient mice (P < 0.001), reflective of plasmid DNA silencing. We then cloned Cre-recombinase in cis on the LoxP-HBV plasmids, achieving plasmid stability in bacteria with intron insertion into Cre and demonstrating rcccDNA formation after transfection in vitro and in vivo. These cis-Cre/LoxP-HBV plasmids were then used to create HBx-mutant and GFP reporter plasmids to further probe cccDNA biology and antiviral strategies against cccDNA. Overall, we believe these auto-generating rcccDNA plasmids will be of great value to model cccDNA for testing new therapies against HBV infection.
Robert L Kruse, Xavier Legras, Mercedes Barzi

2477 related Products with: Cre/LoxP-HBV plasmids generating recombinant covalently closed circular DNA genome upon transfection.

251mg101001mg5100100205010,000IU1mg

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