生物谷报道：Gene Tools公司最新文章描述了一种利用吗啉寡聚核苷酸（Morpholino oligonucleotides，MF）进行miRNA活性敲除、成熟敲除和特异控制的新技术。
microRNA调节基因特别是一大组基因的表达，在胚胎发育、心脏功能、癌症发生等过程中扮演重要角色。miRNA研究的最新发现改变了我们对基因调节的认识，出现了许多miRNA活性的新报道。miRNA活性的实验操作已经成为分子生物学的一种标准工具，但这些技术目前仍处于改进中。
microRNA的成熟过程分为几个阶段。初级miRNA(primary microRNA)转录自DNA，并折叠为一个stem-loop。酶Drosha将stem-loop与转录本(transcript)其它部份分开，接着酶Dicer将loop与stem分开。双链stem与蛋白Argonaute相互作用，Argonaute将双链劈开，释放其中的一条链，与剩下的一条链（guide strand，引导链）形成miRISC复合体。miRISC复合体与mRNA相互作用，改变mRNA的表达。
miRNA的活性敲除通常利用靶向miRNA引导链的寡核苷酸。靶向miRNA的Morpholino oligonucleotides也能干扰miRNA的活性。单独利用这种技术很难控制敲除的特异性。然而，靶向未成熟miRNA的溶核加工位点（nucleolytic processing sites，生物通编者译）的Morpholino，能够抑制miRNA的成熟。如此，靶向初级miRNA的成套的不重叠的Morpholino oligos，可以用于特异控制；如果靶向同一个miRNA的两个不重叠的oligo引发的表型相同，就能说明表型是由于靶miRNA的活性被敲除引起的，而非脱靶效应。这种技术的详细内容刊登于：
Kloosterman WP, Lagendijk AK, Ketting RF, Moulton JD, Plasterk RHA. Targeted inhibition of miRNA maturation with morpholinos reveals a role for miR-375 in pancreatic islet development. PLoS Biol. 2007;5(8): e203.
biology.plosjournals.org/perlserv/?request=get-pdf&file=10.1371_journal.pbio.0050203-L.pdf
已刊载电子版。
GENE TOOLS公司成立于1997年，位于AVI Biopharma公司旗下，是一家生产生物学和医学研究所需的序列个性化的Morpholino oligos的生物技术公司。其Morpholino产品从2000年开始商业化，目前已经有1700篇研究型论文涉及到Morpholino。
GENE TOOLS的初级产品Morpholino反义核苷酸，是合成分子，阻断特定基因活性的Morpholino。Morpholino的亚单位的连接顺序决定了受其阻断的目的基因。GENE TOOLS公司根据客户需要，设计关闭目的基因活性的个性化Morpholino。GENE TOOLS公司通常每周能够合成200多个Morpholino。
GENE TOOLS的客户一般是大学、研究所和制药公司的研究人员。
发育生物学家通过将Morpholino oligos注入卵子或者斑马鱼、青蛙、海鞘、海胆等的受精卵中研究胚胎发育
细胞生物学家利用Morpholino oligos研究新经过测序的基因的功能
医疗工作者利用Morpholino oligos研究癌症等疾病的细胞学机制
想获得关于Morpholino oligos的更多信息，请登陆GENE TOOLS网站（www.gene-tools.com）并阅览文章（en.wikipedia.org/wiki/Morpholino）
原始出处:
PLoS Biology
Received: October 13, 2006; Accepted: May 22, 2007; Published: July 24, 2007
Targeted Inhibition of miRNA Maturation with Morpholinos Reveals a Role for miR-375 in Pancreatic Islet Development
Wigard P. Kloosterman1, Anne K. Lagendijk1, René F. Ketting1*, Jon D. Moulton2, Ronald H. A. Plasterk1
1 Hubrecht Laboratory-KNAW, Utrecht, The Netherlands, 2 Gene Tools, Philomath, Oregon, United States of America
Several vertebrate microRNAs (miRNAs) have been implicated in cellular processes such as muscle differentiation, synapse function, and insulin secretion. In addition, analysis of Dicer null mutants has shown that miRNAs play a role in tissue morphogenesis. Nonetheless, only a few loss-of-function phenotypes for individual miRNAs have been described to date. Here, we introduce a quick and versatile method to interfere with miRNA function during zebrafish embryonic development. Morpholino oligonucleotides targeting the mature miRNA or the miRNA precursor specifically and temporally knock down miRNAs. Morpholinos can block processing of the primary miRNA (pri-miRNA) or the pre-miRNA, and they can inhibit the activity of the mature miRNA. We used this strategy to knock down 13 miRNAs conserved between zebrafish and mammals. For most miRNAs, this does not result in visible defects, but knockdown of miR-375 causes defects in the morphology of the pancreatic islet. Although the islet is still intact at 24 hours postfertilization, in later stages the islet cells become scattered. This phenotype can be recapitulated by independent control morpholinos targeting other sequences in the miR-375 precursor, excluding off-target effects as cause of the phenotype. The aberrant formation of the endocrine pancreas, caused by miR-375 knockdown, is one of the first loss-of-function phenotypes for an individual miRNA in vertebrate development. The miRNA knockdown strategy presented here will be widely used to unravel miRNA function in zebrafish.
