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 Biomics ARCA Cas9 mRNA

      近10年来,mRNA在医学应用领域取得显著进展。研究人员可快速合成任意序列的mRNA片段,并能保存在室温条件下。在无细胞的体系中利用DNA转录生成cap和poly A修饰的mRNA分子稳定性良好,而修饰碱基的mRNA在稳定性上更获得大幅提升。在研究基因敲除动物及其胚胎发育中的基因表达时,科研人员往往都采用卵母细胞的mRNA显微注射的方法,该方法在ZFN、TALEN、CRISPR、重编程细胞多能性等技术中得到广泛应用。在这些基因编辑过程中使用mRNA较之DNA有诸多优点,例如无需进入核内并转录效率更快,RNA易降解也避免基因组遗传重组现象。鉴于此,Biomics Biotech推出mRNA系列产品,提供不同类型的Cas9 mRNA, 例如野生型spCas9 mRNA、单突变spCas9 Nickase mRNA、双突变spCas9 mutant mRNA。此外我们还提供各种报告基因的mRNA给客户作为阳性对照,例如Luciferase mRNA,mCHERRY mRNA, GFP mRβ-gal mRNA

 


 

      Biomics Cas9 mRNA 使用本公司自主开发的T7 cap mRNA MICscript™ KIT转录合成和EzOmics™ RNA Quick Clear Kit纯化,从而使我们的产品成本更低,较客户购买国外同类产品更具性价比。Cas9 mRNA转录DNA模板为线性化质粒,确保无冗余转录序列。该质粒中Cas9基因为人源密码子优化spCas9基因,带有核转导域(NLS)及Poly A信号(globin3’ UTR),Cas9 mRNA在细胞内更具稳定性。

 


 

      细胞注射或转染用mRNA合成还有一个关键步骤是5’端的加帽,帽子结构是指在真核生物中转录后修饰形成的成熟mRNA在5’端的一个特殊结构,即m7GPPPN结构,又称为甲基鸟苷帽子。它是在RNA三磷酸酶,mRNA鸟苷酰转移酶,mRNA(鸟嘌呤-7)甲基转移酶和mRNA(核苷-2’)甲基转移酶催化形成的。mRNA 5’-端帽子结构是mRNA翻译起始的必要结构,对核糖体对mRNA的识别提供了信号,协助核糖体与mRNA结合,使翻译从AUG开始。帽子结构可增加mRNA的稳定性,保护mRNA免遭5’ →3‘核酸外切酶的攻击。Biomics使用抗反向帽子类似物(ARCA) m27,3’-OG[5’]ppp[5’]在体外产生加帽RNA。在体外的转录反应中由于ACRA的m7G核苷上含有一个3’-O甲基,ACRA可仅被整合到RNA5‘端’正确的方向。因此,ARCA的整合导致加帽RNA的合成比标准帽子类似物(mCAP、CAP)翻译更加有效。

 


 

      Biomics mRNA产品浓度为0.5-1μg/μL ,文献报道中显微注射的mRNA使用浓度一般为20-200ng/μL

 


 

FAQ:

  1. In eukaryotes: the 3’ poly(A) tail confers stability.

    A: Most mammalian mRNAs are Polyadenylated

  2. mRNA Decay and Translation

    A: The intimate relationship between mRNA decay and translation is further indicated by the ability of translation-initiation factors (eIF) and proteins (PAB) that bind the poly (A) tail to protect the mRNA from degradation. Moreover, evidence shows that inhibiting translation elongation promotes mRNA stabilization.

  3.  

    Translation initiation complex

     

  4. What is the rate-limiting step in mRNA degradation?

    A: An evolutionarily conserved mRNA-degradation pathway is initiated by the removal of the 3’- poly(A) tail. This disrupts the translation initiation complex and provides degradative enzymes with access to the 5’ cap and remaining RNA body.

Reference

  1. McIvor RS. Therapeutic Delivery of mRNA: The Medium Is the Message,Mol Ther. 2011 May; 19(5):822-3.
  2. Warren et al., Highly Efficient Reprogramming to Pluripotency and Directed Differentiation of Human Cells with Synthetic Modified mRNA, Cell Stem Cell (2010),
  3. Yang H, Wang H, Shivalila CS,Cheng AW, Shi L, Jaenisch R. One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell. 2013 Sep 12; 154(6):1370-9.
  4. Chang N1, Sun C, Gao L, Zhu D, Xu X, Zhu X, Xiong JW, Xi JJ. Genome editing with RNA-guided Cas9 nuclease in Zabrafish embryos. Cell Res. 2013 Apr; 23(4):465-72.
  5. Ma Y, Shen B, Zhang X, Lu Y, Chen W, Ma J, Huang X, Zhang L. Heritable multiplex genetic engineering in rats using CRISPR/Cas9. PLoS One. 2014 Mar 5; 9(3):e89413.
  6. Sung YH, Kim JM,Kim HT,Lee J, Highly efficient gene knockout in mice and Zabrafish with RNA-guided endonuclease. enome Res. 2014 Jan; 24(1):125-31.

关联产品

  1. T7 cap mRNA MICscript™ KIT
  2. EzOmics™ RNA Quick Clear Kit
  3. Reporter mRNA
  4. Custom Long RNA Synthesis Services
  5. Reprogramming mRNA

 

 

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