Alternative approach to generate shRNA from cDNA.
Alternative approach to generate shRNA from cDNA.
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DOI：
10.2144/05384rn02
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发表时间：
2005-04
期刊：
影响因子：
2.7
通讯作者：
Anh Dinh;Y. Mo
中科院分区：
文献类型：
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作者：
Anh Dinh;Y. Mo
基于载体表达合成的短发夹RNA（shRNA）与体外合成的短干扰RNA（siRNA）一样有效地抑制其相应基因的表达。最近，三个研究小组分别报道了一种从cDNA构建shRNA文库的新技术，为在许多生物系统中进行全基因组功能筛选提供了巨大的希望。在本研究中，我们报告了另一种方法，从cDNA产生shRNA。一个主要的改进是使用切口酶打开双链DNA，使得环区保持单链，而DNA片段的其余部分在升高的温度下是双链的（例如，72摄氏度）。然后使用双链区域中存在的链作为引物，通过Taq DNA聚合酶将单链DNA转化为双链DNA。因此，延伸产物携带由环分隔的19 bp的回文结构。最后，将DNA片段克隆到载体中，该载体在上游区域携带H1启动子，并在下游区域以5Ts（Pol III聚合酶的终止子）结束。为了证明这一原理，我们从绿色荧光蛋白（GFP）cDNA中构建了短发夹RNA，并成功抑制了GFP的表达。因此，这种简化的方法提供了一种更好的替代方案，从cDNA产生shRNA文库。这样的shRNA文库可用于通过多种选择方法鉴定潜在的siRNA靶序列和研究基因功能。
Short hairpin RNA (shRNA) synthesized from vector-based expression is as effective as short interfering RNA (siRNA) synthesized in vitro for suppressing the expression of their corresponding genes. Recently, three groups independently reported a new technology to construct an shRNA library from cDNA, providing great hope for genome-wide functional screens in many biological systems. In the present study, we report an alternative approach to generate shRNA from cDNA. A major improvement was to use a nicking enzyme to open up the double-stranded DNA so that the loop region remains single-stranded while the rest of the DNA fragment is double-stranded at an elevated temperature (e.g., 72 degrees C). The single-stranded DNA was then converted into double-stranded DNA by Taq DNA polymerase using the existing strand in the double-stranded region as a primer. Thus, the extended product carried a palindromic structure of 19 bp separated by a loop. Finally, the DNA fragment was cloned into a vector that carries an H1 promoter at the upstream region and ends with 5Ts, a terminator for the Pol III polymerase, at the downstream region. To prove the principle, we constructed shRNA from green fluorescent protein (GFP) cDNA and successfully suppressed GFP expression. Consequently, this simplified approach provides a better alternative to generate shRNA libraries from cDNA. Such shRNA libraries can be used to identify potential siRNA target sequences and study gene functions by a variety of selection methods.