Research using hematopoietic cells and other suspension cells has been limited in part by low efficiency of gene transfer (transduction and transfection) into these cell types. RetroNectin reagent promotes colocalization of lentivirus or retrovirus with target cells to dramatically enhance transduction efficiency. As a consequence, RetroNectin reagent is now the gold standard transduction enhancer for retroviral/lentiviral gene transfer to hematopoietic cells and holds great promise in clinical application, having been used for over 40 clinical trials.
Research using hematopoietic cells and other suspension cells has been limited in part by low efficiency of gene transfer (transduction and transfection) into these cell types. RetroNectin reagent promotes colocalization of lentivirus or retrovirus with target cells to dramatically enhance transduction efficiency. As a consequence, RetroNectin reagent is now the gold standard transduction enhancer for retroviral/lentiviral gene transfer to hematopoietic cells and holds great promise in clinical application, having been used for over 40 clinical trials.
What is RetroNectin reagent?
RetroNectin reagent is a recombinant human fibronectin fragment (rFN-CH-296) that contains three functional domains: the cell-binding domain, the heparin-binding domain, and the CS-1 sequence.
How does RetroNectin reagent work?
RetroNectin enhances lentiviral- and retroviral-mediated gene transduction by aiding the colocalization of target cells and viral particles. Specifically, virus particles bind RetroNectin reagent via interaction with the H-domain, and target cells bind mainly through the interaction of cell surface integrin receptor VLA-5 and VLA-4 with the fibronectin C-domain and CS-1 site, respectively. By facilitating close physical proximity, RetroNectin reagent can enhance viral-mediated gene transfer to target cells expressing integrin receptors VLA-4 and/or VLA-5. VL-4-expressing cells include T cells, B cells, monocytes, NK cells, eosinophils, bone marrow monocytic cells, and lymphoid progenitors. Thymocytes, activated T cells, and mast cells express VLA-5.
RetroNectin reagent also can be used to enhance T-cell expansion (CD8+ cells) in vitro
T-lymphocyte expansion from peripheral blood mononuclear cells (PBMCs) is usually performed in the presence of interleukin-2 (IL-2) and anti-CD3 antibody stimulation. The efficiency of T-cell expansion can be significantly increased in the presence of RetroNectin reagent. Moreover, the T-cell population after expansion in the presence of RetroNectin reagent contains a high proportion of naive Tcells.
FAQs regarding proper use of RetroNectin GMP grade
- Question: Can I use Takara Bio"s RetroNectin GMP grade in clinical trials?Answer: Researchers are free to use this product for development of research-based clinical trials. Proper regulatory approval is required before using the GMP-grade product in research-based clinical trials since these trials may require special considerations ormore stringentmanufacturing standards.
- Question: Can I use Takara Bio"s RetroNectin GMP grade for commercial use?Answer: Researchers are free to use the product in accordance with the Notice to Purchaser, but commercial use is not allowed under our general Terms and Conditions for purchase of these products. Commercial access to the GMP-grade product may be available through a formal License and Supply Agreement. In all cases, it is the researcher’s responsibility to get approval from the appropriate regulatory authorities for the intended use of these ancillary materials for your application.
- Question: Can I use RetroNectin GMP grade for in vivo clinical use?Answer: No. The product cannot be used for direct in vivo applications. The product is intended for use as an ancillary reagent in a manufacturing process which includes other steps prior to any in vivo application.
- Question: Is RetroNectin GMP grade (#T202) registered in DMF?Answer: A DMF (18898) was submitted for the liquid format RetroNectin GMP grade on March 15, 2019.
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逆转录（reverse transcription）是以RNA为模板合成DNA的过程，即RNA指导下的DNA合成。此过程中，核酸合成与转录（DNA到RNA）过程与遗传信息的流动方向（RNA到DNA）相反，故称为逆转录。逆转录过程是RNA病毒的复制形式之一，需逆转录酶的催化。 逆转录过程的揭示是分子生物学研究中的重大发现，是对中心法则的重要修正和补充。人们通过体外模拟该过程，以样本中提取的mRNA为模板，在逆转录酶的作用下，合成出互补的cDNA，构建cDNA文库，并从中筛选特异的目的基因。该方法已成为基因工程技术中最常用的获得目的基因的策略之一。
1.模板提取（一般为RNA）：Trizol、氯仿、异丙醇、无水乙醇、DEPC处理水
2.模板浓度测定：分光光度计或NanoDrop
3.逆转录：逆转录试剂盒（或者一步法试剂盒），这一步可以用普通PCR做，也可以用水域做。
4.荧光定量PCR试剂：通常有用SYBR Green Mix做的，但是这里建议你用EvaGreen做，灵敏度和平行性都要好于SYBR Green，并且如果你那是ABI或者Stratagene的PCR如果用SYBR Green还需要加一步Rox很麻烦。
5.其他：除了以上的那些还需要离心管、PCR管或板（Axygen反应比较好）、移液枪等，暂时就想到这么多。
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大家miRNA逆转录和qPCR的试剂盒用的是那个公司的啊，求推荐！
