A CRISPR toolbox for generating intersectional genetic mouse models for functional, molecular, and anatomical circuit mapping

In addition to single recombinase systems, the expression of two recombinases in distinct, but partially overlapping, populations allows for more defined target expression. Although the application of this method is becoming increasingly popular, its experimental implementation has been broadly restricted to manipulations of a limited set of common alleles that are often commercially produced at great expense, with costs and technical challenges associated with the production of intersectional mouse lines hindering customized approaches to many researchers Joblinks Human ADAM10 cDNA. Here, we present a simplified CRISPR toolkit for rapid, inexpensive, and facile intersectional allele production.
Results: Briefly, we produced 7 intersectional mouse lines using a dual recombinase system, one mouse line with a single recombinase system, and three embryonic stems (ES) cell lines that are designed to study the way functional, molecular, and anatomical features relate to each other in building circuits that underlie physiology and behavior.
As a proof-of-principle, we applied three of these lines to different neuronal populations for anatomical mapping and functional in vivo investigation of respiratory control.
We also generated a mouse line with a single recombinase-responsive allele that controls the expression of the calcium sensor Twitch-2B. This mouse line was applied globally to study the effects of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) on calcium release in the ovarian follicle.
Conclusions: The lines presented here are representative examples of outcomes possible with the successful application of our genetic toolkit for the facile development of diverse, modifiable animal models. This toolkit will allow labs to create single or dual recombinase effector lines easily for any cell population or subpopulation of interest when paired with the appropriate Cre and FLP recombinase mouse lines or viral vectors. We have made our tools and derivative intersectional mouse and ES cell lines openly available for non-commercial use through publicly curated repositories for plasmid DNA, ES cells, and transgenic mouse lines.

An enhanced method for nucleic acid detection with CRISPR-Cas12a using phosphorothioate modified primers and optimized gold-nanopaticle strip

CRISPR-Cas12a system has been shown promising for nucleic acid diagnostics due to its rapid, portable and accurate features. However, cleavage of the amplicons and primers by the cis– and trans-activity of Cas12a hinders the attempts to integrate the amplification and detection into a single reaction. Through phosphorothioate modification of primers, we realized onepot detection with high sensitivity using plasmids of SARS-CoV-2, HPV16 and HPV18. We also identified the activated Cas12a has a much higher affinity to C nucleotide-rich reporter than others.
By applying such reporters, the reaction time required for a lateral-flow readout was significantly reduced. Furthermore, to improve the specificity of the strip-based assay, we created a novel reporter and, when combined with a customized gold-nanopaticle strip, the readout was greatly enhanced owing to the elimination of the nonspecific signal.
This established system, termed Targeting DNA by Cas12a-based Eye Sight Testing in an One-pot Reaction (TESTOR), was validated using clinical cervical scrape samples for human papillomaviruses (HPVs) detection. Our system represents a general approach to integrating nucleic acid amplification and detection into a single reaction in CRISPR-Cas systems, highlighting its potential as a rapid, portable and accurate detection platform of nucleic acids.

Detection of plasmid contigs in draft genome assemblies using customized Kraken databases

Plasmids play an important role in bacterial evolution and mediate horizontal transfer of genes including virulence and antimicrobial resistance genes. Although short-read sequencing technologies have enabled large-scale bacterial genomics, the resulting draft genome assemblies are often fragmented into hundreds of discrete contigs. Several tools and approaches have been developed to identify plasmid sequences in such assemblies, but require trade-off between sensitivity and specificity. Here we propose using the Kraken classifier, together with a custom Kraken database comprising known chromosomal and plasmid sequences of Klebsiella pneumoniae species complex (KpSC), to identify plasmid-derived contigs in draft assemblies.
We assessed performance using Illumina-based draft genome assemblies for 82 KpSC isolates, for which complete genomes were available to supply ground truth. When benchmarked against five other classifiers (Centrifuge, RFPlasmid, mlplasmids, PlaScope and Platon), Kraken showed balanced performance in terms of overall sensitivity and specificity (90.8 and 99.4 %, respectively, for contig count; 96.5 and >99.9 %, respectively, for cumulative contig length), and the highest accuracy (96.8% vs 91.8-96.6% for contig count; 99.8% vs 99.0-99.7 % for cumulative contig length), and F1-score (94.5 % vs 84.5-94.1 %, for contig count; 98.0 % vs 88.9-96.7 % for cumulative contig length). Kraken also achieved consistent performance across our genome collection. Furthermore, we demonstrate that expanding the Kraken database with additional known chromosomal and plasmid sequences can further improve classification performance. Although we have focused here on the KpSC, this methodology could easily be applied to other species with a sufficient number of completed genomes.

Exploiting heterologous and endogenous CRISPR-Cas systems for genome editing in the probiotic Clostridium butyricum

Clostridium butyricum has been widely used as a probiotic for humans and food animals. However, the mechanisms of beneficial effects of C. butyricum on the host remain poorly understood, largely due to the lack of high-throughput genome engineering tools. Here, we report the exploitation of heterologous Type II CRISPR-Cas9 system and endogenous Type I-B CRISPR-Cas system in probiotic C. butyricum for seamless genome engineering. Although successful genome editing was achieved in C. butyricum when CRISPR-Cas9 system was employed, the expression of toxic cas9 gene result in really poor transformation, spurring us to develop an easy-applicable and high-efficient genome editing tool.
Therefore, the endogenous Type I-B CRISPR-Cas machinery located on the megaplasmid of C. butyricum was co-opted for genome editing. In vivo plasmid, interference assays identified that ACA and TAA were functional protospacer adjacent motif (PAM) sequences needed for site-specific CRISPR attacking. Using the customized endogenous CRISPR-Cas system, we successfully deleted spo0A and aldh genes in C. butyricum, yielding an efficiency of up to 100%.
Moreover, the conjugation efficiency of endogenous CRISPR-Cas system was dramatically enhanced due to the precluding expression of cas9. Altogether, the two approaches developed herein remarkably expand the existing genetic toolbox available for investigation of C. butyricum. This article is protected by copyright. All rights reserved.
ADAM10 (Human)
GT15078 100 ug 631.2 EUR
KT-5207 96 tests 952 EUR
Human ADAM10 Protein
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Human ADAM10 Protein
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Human ADAM10 Protein
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Human ADAM10 Protein
abx262305-5mg 5 mg 225 EUR
ADAM10 (Human), CF
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ADAM10 (untagged)-Human ADAM metallopeptidase domain 10 (ADAM10)
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ADAM10 siRNA (Human)
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ADAM10 siRNA (Human)
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Delivery of superoxide dismutase by TAT and Abalone peptides for the protection of skin cells against oxidative stress

This work aimed to clone, express, purify and evaluate the protective effect antioxidant of this enzyme on skin cells when fused to transactivator of transcription (TAT) protein transduction domain of HIV-1 and Abalone (Ab) peptides to allow cell penetration. TrSOD, TAT-TrSOD-Yfp (fused to yellow fluorescent protein) and Ab-TrSOD were expressed in E.coli and purified as soluble proteins. The cytotoxicity of the enzymes, at the concentrations of 1, 3 and 6 μmol/L, was evaluated for a period of 24 and 48 h of incubation, with no cytotoxic effect on 3T3 fibroblasts. The 3T3 cells were exposed to the oxidant agent tert-butyl hydroperoxide (tBH) and evaluated for ROS generation, in the presence or not of the recombinant enzymes.
TAT-TrSOD-Yfp was able to decrease the generation of ROS in 15% when used in the concentrations of 3 and 6 μmol/L in comparison to the control, but there was no difference in relation to the effect of TrSOD. Ab-TrSOD, when compared to TrSOD, promoted a decrease in the formation of ROS of 19 and 14% at the concentrations of 1 and 6 μmol/L, respectively, indicating that this joplink Recombinant Human Regulator of G-protein form was more effective in reducing oxidative stress compared to SOD without the cell penetrating peptide (CPP).
Together, these results indicate that the fusion of SOD with these CPP increased the antioxidant capacity of fibroblasts, identified by the reduction in the generation of ROS. In addition, such molecules, in the concentrations initially used, were not toxic to the cells, opening perspectives for the development of products for antioxidant protection of the skin that may have therapeutic and cosmetic application. This article is protected by copyright.

Comparative Immunogenicity of the Recombinant Receptor-Binding Domain of Protein S SARS-CoV-2 Obtained in Prokaryotic and Mammalian Expression Systems

The receptor-binding domain (RBD) of the protein S SARS-CoV-2 is considered to be one of the appealing targets for developing a vaccine against COVID-19. The choice of an expression system is essential when developing subunit vaccines, as it ensures the effective synthesis of the correctly folded target protein, and maintains its antigenic and immunogenic properties. Here, we describe the production of a recombinant RBD protein using prokaryotic (pRBD) and mammalian (mRBD) expression systems, and compare the immunogenicity of prokaryotic and mammalian-expressed RBD using a BALB/c mice model.
An analysis of the sera from mice immunized with both variants of the protein revealed that the mRBD expressed in CHO cells provides a significantly stronger humoral immune response compared with the RBD expressed in E.coli cells. A specific antibody titer of sera from mice immunized with mRBD was ten-fold higher than the sera from the mice that received pRBD in ELISA, and about 100-fold higher in a neutralization test. The data obtained suggests that mRBD is capable of inducing neutralizing antibodies against SARS-CoV-2.

Preparation of highly specific monoclonal antibodies against SARS-CoV-2 nucleocapsid protein and the preliminary development of antigen detection test strips

The coronavirus disease 2019 (COVID-19) are outbreaking all over the world. To help fight this disease, it is necessary to establish an effective and rapid detection method. The nucleocapsid (N) protein of Severe Acute Respiratory syndrome Coronavirus 2 (SARS-CoV-2) is involved in viral replication, assembly and immune regulation and plays an important role in the viral life cycle. Moreover, the N protein also could be a diagnostic factor and potential drag target.
Therefore, by synthesizing the N gene sequence of SARS-CoV-2, constructing the pET-28a (+)-N recombinant plasmid, we expressed the N protein in E.coli and obtained 15 mAbs against SARS-CoV-2-N protein by the hybridomas and ascites, then an immunochromatographic test strip method detecting N antigen was established.
In this study, we obtained 14 high-titer and high-specificity monoclonal antibodies, and the test strips exclusively react with the SARS-CoV-2-N protein and no cross-reactivity with other coronavirus and also recognize the recombinant N protein of Delta (B.1.617.2) variant. These mAbs can be used for the early and rapid diagnosis of SARS-CoV-2 infection through serological antigen. This article is protected by copyright. All rights reserved.

Preparation and identification of rat polyclonal antibody against SARS-CoV-2 main protease (Mpro)

Objective To investigate the immunological functions of SARS-CoV-2 main protease (Mpro) in coronavirus disease 2019 (COVID-19), polyclonal antibody against Mpro was developed. Methods A codon-optimized SARS-CoV-2 Mpro gene was synthesized and ligated into a pET-28a vector for construction of a recombinant plasmid named by pET-28a-Mpro. Subsequently, this plasmid was transformed into E.coli Rosetta (DE3) competent cells for Mpro expression in an optimized condition, and then Mpro was purified using a HisTrap chelating column.
The purified Mpro was used as immunogen to inoculate rats and the serum was collected after third immunization cycle. The titer, selectivity and sensitivity of polyclonal antibody against Mpro were analyzed using the ELISA and Western blot analysis. Results An optimized expression condition in E.coli cells for Mpro was determined, and the recombinant Mpro was purified by a HisTrap chelating column. The ELISA and Western blot analysis demonstrated that the highly sensitive polyclonal antibody against Mpro specially recognized the recombinant Mpro, and the titer reached 1:256 000. Conclusion The highly specific polyclonal antibody against SARS-CoV-2 Mpro is successfully prepared, which lays an experimental foundation for investigating the immunological function of Mpro in COVID-19.

Direct enzyme-linked aptamer assay (DELAA) for diagnosis of toxoplasmosis by detection of SAG1 protein in mice and humans

Toxoplasma gondii is a single-celled parasite commonly found in mammals and birds. Diagnosis of toxoplasmosis largely depends on measurements of the antibody and/or antigen and Toxoplasma DNAs due to the presence of tissue dwelling duplicating tachyzoites, or quiescent cysts in latent infection of the parasite. As a major surface antigen of T.gondii tachyzoites, SAG1 is a key molecule for laboratory diagnosis. However, there are no methods available yet for SAG1 detection using aptamer-based technology. Recombinant SAG1 (r-SAG1) of Toxoplasma WH3 strain (type Chinese 1) was expressed in E.coli and subjected to the synthetic oligonucleotide library for selection of nucleic acid aptamers which target the r-SAG1 antigen, with systematic evolution of ligands by exponential enrichment (SELEX) strategy.
The specific aptamers were screened out and used in direct enzyme-linked aptamer assay (DELAA) for detection of native SAG1 (n-SAG1) obtained from tachyzoite lysates, mouse sera of acute infection, and human sera that had been verified for Toxoplasma DNAs by PCR amplification. As results, the soluble r-SAG1 protein was obtained from E.coli lysates by purification and identification with immunoblotting, followed by biotinylation. The selected aptamers were amplified by PCR and DNA sequencing.
The results showed that the aptamer-2, with the highest affinity to n-SAG1 in the sera of animals with minimal difference in the four aptamer candidates, has a high specificity and sensitivity when used in detection of n-SAG1 in the sera of humans when compared with the commercial kit of ELISA for T.gondii circulating antigen test. We concluded that a new direct enzyme-linked aptamer assay (DELAA) was developed for the detection of the n-SAG1 protein of T. gondii. With increased sensitivity and specificity, stability, easy and cheap preparation, the aptamer-based technology is considered an efficient method for the diagnosis of active as well as reactivated toxoplasmosis.
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Recombinant Human SLC2A4 regulator (SLC2A4RG)
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Recombinant Human SLC2A4 regulator (SLC2A4RG)
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