National Animal Protozoa Laboratory, Key Laboratory of Zoonosis of the Ministry of Agriculture, College of Veterinary Medicine, China Agriculture University, Beijing, China.
Biotechniques. 2012 Mar;52(3):173-6. doi: 10.2144/000113821.
The nematode Caenorhabditis elegans is an important model animal for biological research. Currently, transgenic C. elegans strains are mainly generated by injecting DNA encoding a gene of interest, in combination with a reporter gene, into the gonad. With this approach, the interpretation of negative results, such as the failure to observe reporter expression, is frequently required. Single, selectable vectors are urgently required. Internal ribosome entry site (IRES) elements are known to bind the eukaryotic ribosomal translation initiation complex and independently promote translation initiation. Bioinformatic analysis predicted an IRES motif upstream of the start codon of the C. elegans Hsp-3 gene. While this sequence has a Y-shaped double-hairpin secondary structure characteristic of IRES elements, it was unclear if it could function as an IRES. In the present study, this predicted Hsp-3 IRES was incorporated into a bicistronic vector driven by the myo-3 promoter, which allowed co-expression of RFP and GFP genes in the muscle tissue of C. elegans and thereby demonstrated that this IRES element is functional. This vector provides a novel, powerful tool for C. elegans research.
秀丽隐杆线虫是生物学研究中的一种重要模式动物。目前，转基因秀丽隐杆线虫株主要通过将目的基因的 DNA 编码与报告基因一起注射到性腺中来产生。使用这种方法，经常需要解释阴性结果，例如未能观察到报告基因的表达。因此迫切需要单可选择的载体。内部核糖体进入位点 (IRES) 元件已知能结合真核核糖体翻译起始复合物，并独立地促进翻译起始。生物信息学分析预测秀丽隐杆线虫 HSP-3 基因起始密码子上游存在一个 IRES 基序。虽然该序列具有 IRES 元件的 Y 形双链发夹二级结构特征，但尚不清楚它是否能作为 IRES 发挥作用。在本研究中，该预测的 HSP-3 IRES 被整合到一个由肌球蛋白-3 启动子驱动的双顺反子载体中，该载体允许在秀丽隐杆线虫的肌肉组织中共同表达 RFP 和 GFP 基因，从而证明该 IRES 元件是有功能的。该载体为秀丽隐杆线虫研究提供了一种新颖而强大的工具。
