Takara 6144

[Fritzi Vanderweel]

TakaraIVTpro mRNA Synthesis System offers a complete solution for synthesizing high-quality single-stranded mRNA from your gene of interest, with a cap structure (using CleanCap Reagent AG from TriLink Biotechnology or similar, not included) and poly(A) sequence. The Takara IVTpro mRNA Synthesis System (Cat. # 6141) comprises:

Takara IVTpro mRNA Synthesis System includes all components necessary from DNA template preparation to high-yield in vitro transcription reaction. Easily scale up to 10-fold (200 μl reaction volume) without affecting the expected mRNA yield. This kit includes the Cloning Kit for mRNA Template (Cat. # 6143) and Takara IVTpro mRNA Synthesis Kit (Cat. # 6144), which are also sold separately.

This product is for in vitro diagnostic use. Takara products may not be resold or transferred, modified for resale or transfer, or used to manufacture commercial products without written approval from Takara Bio Inc. If you require licenses for other use, please contact us by phone at +81 77 .565. 6976 or from our website at -bio.co.jp/inquiry/ivd/. Your use of this product is also subject to compliance with any applicable licensing requirements described on the product web page. It is your responsibility to review, understand and adhere to any restrictions imposed by such statements. All trademarks are the property of their respective owners. Certain trademarks may not be registered in all jurisdictions.

The mRNA yield was proportional to the reaction volume up to 200 l with no significant change in mRNA concentration (Panel A). Scaling up of the IVT reaction volume does not affect the quality of RNA product (Panel B).

Purification of RNA product using LiCl was comparable to purification with NucleoSpin column when two elutions were performed (Panel A). HEK293T cells transfected with mRNA purified using LiCl shows protein expression comparable to mRNA purified using NucleoSpin columns (Panel B).

The IVT reaction was performed using various sizes of DNA Templates with CleanCap Reagent AG (3' OMe) and N1-methyl pseudo UTP. Each resulting mRNA (200 ng) was denatured at 65 C for 10 min, followed by analysis using 1.2% formaldehyde gel electrophoresis.

Cloning Kit for mRNA Template enables easy cloning of a template DNA into a pre-linearized plasmid (included in the kit) for downstream in vitro transcription reactions. The pre-linearized plasmid contains the T7 promoter, transcription initiation sequence (AGG), 5'- and 3'-UTRs (untranslated region), and poly(A) sequence. The kit also includes an In-Fusion Snap Assembly Master Mix for seamless cloning of the target DNA into the pre-linearized vector and a FLuc Control Fragment, consisting of 15 bp 3' and 5' In-Fusion sequences and Fluc CDS (1,683 bp).

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

Takara IVTpro mRNA Synthesis Kit enables the production of large amounts of mRNA through in vitro transcription using an optimized, highly efficient T7 RNA polymerase. The kit includes separate NTPs for easy optimization or replacement with modified nucleosides, such as pseudouridine. The kit synthesizes up to 200 μg or more of mRNA per 20 μl reaction, includes DNase I to digest the template DNA after the in vitro transcription reaction and a lithium chloride (LiCl) solution to purify the synthesized mRNA for downstream applications.

Please see the product's Certificate of Analysis for information about storage conditions, product components, and technical specifications. Please see the Kit Components List to determine kit components. Certificates of Analysis and Kit Components Lists are located under the Documents tab.

Takara Bio Europe is a member of the Takara Bio Group, a leading life sciences company that is committed to improving the human condition through biotechnology. Through our Takara, Clontech, and Cellartis brands, our mission is to develop high-quality innovative tools and services to accelerate discovery.

What does it take to generate good science? Careful planning, dedicated researchers, and the right tools. At Takara Bio, we thoughtfully develop best-in-class products to tackle your most challenging research problems, and have an expert team of technical support professionals to help you along the way, all at superior value.

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The transcription factor IRF5 has been implicated as a therapeutic target for the autoimmune disease systemic lupus erythematosus (SLE). However, IRF5 activation status during the disease course and the effects of IRF5 inhibition after disease onset are unclear. Here, we show that SLE patients in both the active and remission phase have aberrant activation of IRF5 and interferon-stimulated genes. Partial inhibition of IRF5 is superior to full inhibition of type I interferon signaling in suppressing disease in a mouse model of SLE, possibly due to the function of IRF5 in oxidative phosphorylation. We further demonstrate that inhibition of IRF5 via conditional Irf5 deletion and a newly developed small-molecule inhibitor of IRF5 after disease onset suppresses disease progression and is effective for maintenance of remission in mice. These results suggest that IRF5 inhibition might overcome the limitations of current SLE therapies, thus promoting drug discovery research on IRF5 inhibitors.

Systemic lupus erythematosus (SLE) is an intractable chronic autoimmune disease characterized by a breakdown of immune tolerance to nuclear self-antigens1,2. The patients are primarily females of reproductive age and manifest the production of autoantibodies, such as those against double-stranded DNA (dsDNA) and ribonucleoproteins. Immune complexes formed by these autoantibodies are deposited in various organs and induce recurrent inflammatory lesions. There are genetic, environmental (infection and sunburn), and hormonal (estrogen) factors that influence the disease development3.

One of the highly anticipated new drugs currently in clinical trials is an anti-type I interferon (IFN) receptor monoclonal antibody that blocks the action of type I IFNs (IFN-α and -β)9. SLE patients display high expression of IFN-stimulated genes (ISGs) called the IFN signature10, and animal experiments have revealed that blocking of type I IFN signaling inhibits SLE-like symptoms11,12,13. Recently, it was announced that phase III trials of anifrolumab, an antagonist antibody targeting anti-IFN-α and -β receptor subunit 1 (IFNAR1), reached their primary endpoint14. The percentage of responders was higher in the anifrolumab group (47.8%) than in the placebo group (31.5%). Nevertheless, the annualized relapse rate was still as high as 43% in the anifrolumab group. Thus, the development of additional therapies other than (or in addition to) blocking of type I IFN signaling is desirable.

The main focus of SLE research has been the adaptive immune system, e.g., aberrant activation of autoreactive T and B cells. Currently, however, it is acknowledged that the innate immune system, which initiates inflammation and actuates the adaptive immune system, also significantly contributes to the disease pathogenesis15,16. Indeed, multiple genome-wide association studies have identified IRF5 as one of the genes whose genetic variants are highly associated with SLE risk17,18; IRF5 encodes a transcription factor called IFN regulatory factor 5, which positively regulates endosomal Toll-like receptor (TLR)-mediated, myeloid differentiation primary response protein 88 (MyD88)-dependent innate immune responses19,20. Stimulation of TLR7, TLR8, or TLR9 activates IRF5 via its phosphorylation and nuclear translocation, leading to the induction of type I IFN and inflammatory cytokine genes21,22. IRF5 has been shown to be activated in monocytes of most SLE patients, and its expression is further induced by type I IFNs and estrogen23,24. Moreover, we and other laboratories have demonstrated that even a half IRF5 deficiency prevents disease onset in various mouse models of SLE25,26,27,28. IRF5 functions in multiple cell types involved in SLE pathogenesis, for example, conventional dendritic cells (cDCs), plasmacytoid DCs (pDCs), follicular DCs, monocytes, and B cells17,21. Therefore, IRF5 appears to be a key factor of various steps of SLE pathogenesis, despite the heterogeneous nature of the disease.

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