Search
Search
查看更多产品信息 BLOCK-iT™ Pol II miR-LMNA Validated miRNA Control Vector - FAQs (V4935200)
8 个常见问题解答
该载体可用于稳定表达并且能够使用病毒导入。该miR RNAi载体包括内源性 miRNA 的侧翼序列和茎环序列，可指导从较长的Pol lI转录本（pri-miRNA）上切除改造过的miRNA。当存在细胞核时，这些载体可有效使用内源性细胞器加工生成具有敲低作用的序列，这些经过特殊设计的序列与您的目标序列具有100%同源性，可引起靶标切割。此外，茎环序列具有特殊的限制性酶切位点，因此，它能够实现线性化而获得更有效的测序，这对具有发夹结构的标准的shRNA有时是一个挑战。使用该试剂盒可获得超过70%的敲低成功率，轻松追踪表达（可共同表达绿色荧光蛋白），实现同时敲低多个靶标以及组成型或诱导型表达。
These vectors can be used for stable expression and the ability to use viral delivery. These miR RNAi vectors include flanking and loop sequences from an endogenous miRNA which directs the excision of the engineered miRNA from a longer Pol II transcript (pri-miRNA). When present in the nucleus, these vectors efficiently use the endogenous cellular machinery to process knockdown sequences that are specifically designed to have 100% homology to your target of interest and will result in target cleavage. In addition, the loop sequence has a unique restriction site, so that it can be linearized for more efficient sequencing, sometimes a challenge with standard shRNA hairpins. The kits offer over 70% knockdown success, easy expression tracking (with co-cistronic expression of Green Fluorescent Protein), multiple target knockdown, and constitutive or inducible expression.
The BLOCK-iT miR RNAi expression system allows you to take advantage of promoter flexibility by choosing from a variety of Pol II promoters like CMV, Ubc, tissue specific, or inducible promoters. The miRNA vectors also allow you to clone multiple sequences in the same vector, thereby enabling you to target more than one gene or more than one location in a gene using a single plasmid. An additional advantage offered by some of the miRNA expression vectors is that transfection efficiency can be monitored with the EmGFP fusion partner.
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.
Yes. The miRNA cloning vectors are designed such that you can easily chain together multiple miRNAs to express them in one transcript. This can be used to increase transcription levels of the same miRNA sequence, or to combine multiple different sequences in one co-cistronic expression cassette.
Yes. The miR miRNA vectors are Gateway cloning compatible, and you could use Gateway cloning to transfer the miR miRNA expression cassette to any of our Gateway-adapted viral expression vectors.
The miRNA vectors use endogenous miRNA processing machinery to allow targeted RNAi knockdown of specific genes, but they are not tested for use in the analysis of endogenous miRNA. Endogenous miRNAs are typically not 100% homologous to their targets and thus invoke translational suppression. Our products are designed with 100% homology to the target gene and generally result in target mRNA cleavage.
Both systems are used for gene targeting or gene knockdown but each has distinctive features. The shRNA expression vectors like pENTR/U6 or pENTR/H1-TO use Pol III promoters, whereas the miRNA expression vectors are flexible to use more common and more processive Pol II promoters like CMV, EF1 or other mammalian expression promoters. You can only clone a single shRNA sequence into an shRNA vector to target a single gene, whereas multiple miRNA sequences can be cloned together into an miRNA vector to target one or more genes, or multiple locations in a gene. An additional feature of the miRNA expression vectors is that, due to use of Pol II promoters, the miRNA can be expressed directly in fusion with a reporter gene like EmGFP to monitor transfection and transcription.
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.
All the BLOCK-iT miR RNAi expression vectors are Gateway-adapted and contain the CMV promoter. If more specialized expression is required with a different promoter, the miRNA vectors allow for easy recombination with any other suitable destination vector that Thermo Fisher Scientific offers. A wide variety of Gateway Destination vectors that contain promoters such as EF-1apha or Ubc are available, and all be used for miRNA expression. MultiSite Gateway Technology vectors are also available which will enable you to use a tissue-specific promoter or a promoter of your choice to express miRNA.