生物谷報道：Gene Tools公司最新文章描述了一種利用嗎啉寡聚核苷酸（Morpholino oligonucleotides,，MF）進行miRNA活性敲除,、成熟敲除和特異控制的新技術(shù)。
microRNA調(diào)節(jié)基因特別是一大組基因的表達,，在胚胎發(fā)育,、心臟功能、癌癥發(fā)生等過程中扮演重要角色,。miRNA研究的最新發(fā)現(xiàn)改變了我們對基因調(diào)節(jié)的認(rèn)識,，出現(xiàn)了許多miRNA活性的新報道。miRNA活性的實驗操作已經(jīng)成為分子生物學(xué)的一種標(biāo)準(zhǔn)工具,，但這些技術(shù)目前仍處于改進中,。
microRNA的成熟過程分為幾個階段。初級miRNA(primary microRNA)轉(zhuǎn)錄自DNA,，并折疊為一個stem-loop,。酶Drosha將stem-loop與轉(zhuǎn)錄本(transcript)其它部份分開，接著酶Dicer將loop與stem分開,。雙鏈stem與蛋白Argonaute相互作用,，Argonaute將雙鏈劈開，釋放其中的一條鏈,，與剩下的一條鏈（guide strand,，引導(dǎo)鏈）形成miRISC復(fù)合體。miRISC復(fù)合體與mRNA相互作用,，改變mRNA的表達,。
miRNA的活性敲除通常利用靶向miRNA引導(dǎo)鏈的寡核苷酸。靶向miRNA的Morpholino oligonucleotides也能干擾miRNA的活性,。單獨利用這種技術(shù)很難控制敲除的特異性,。然而，靶向未成熟miRNA的溶核加工位點（nucleolytic processing sites,，生物通編者譯）的Morpholino,，能夠抑制miRNA的成熟。如此,，靶向初級miRNA的成套的不重疊的Morpholino oligos,，可以用于特異控制；如果靶向同一個miRNA的兩個不重疊的oligo引發(fā)的表型相同,，就能說明表型是由于靶miRNA的活性被敲除引起的,，而非脫靶效應(yīng),。這種技術(shù)的詳細(xì)內(nèi)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
已刊載電子版。
GENE TOOLS公司成立于1997年,，位于AVI Biopharma公司旗下,，是一家生產(chǎn)生物學(xué)和醫(yī)學(xué)研究所需的序列個性化的Morpholino oligos的生物技術(shù)公司。其Morpholino產(chǎn)品從2000年開始商業(yè)化,，目前已經(jīng)有1700篇研究型論文涉及到Morpholino,。
GENE TOOLS的初級產(chǎn)品Morpholino反義核苷酸，是合成分子,，阻斷特定基因活性的Morpholino,。Morpholino的亞單位的連接順序決定了受其阻斷的目的基因。GENE TOOLS公司根據(jù)客戶需要,，設(shè)計關(guān)閉目的基因活性的個性化Morpholino,。GENE TOOLS公司通常每周能夠合成200多個Morpholino。
GENE TOOLS的客戶一般是大學(xué),、研究所和制藥公司的研究人員,。
發(fā)育生物學(xué)家通過將Morpholino oligos注入卵子或者斑馬魚、青蛙,、海鞘,、海膽等的受精卵中研究胚胎發(fā)育
細(xì)胞生物學(xué)家利用Morpholino oligos研究新經(jīng)過測序的基因的功能
醫(yī)療工作者利用Morpholino oligos研究癌癥等疾病的細(xì)胞學(xué)機制
想獲得關(guān)于Morpholino oligos的更多信息，請登陸GENE TOOLS網(wǎng)站（www.gene-tools.com）并閱覽文章（en.wikipedia.org/wiki/Morpholino）
原始出處:
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.
