Kaur Gursharan, Lohia Anuradha
Department of Biochemistry, Bose Institute, Kolkata, India.
Biochem Biophys Res Commun. 2004 Aug 6;320(4):1118-22. doi: 10.1016/j.bbrc.2004.06.064.
In order to inhibit gene expression in Entamoeba histolytica, we have developed a method based on expressing double strand RNA interference constructs in stable transformants. The 5' end of Eh Dia was cloned head to head with an intervening non-specific stuffer fragment in the E. histolytica expression vector pJST4. This construct was transformed in E. histolytica HM1:IMSS trophozoites and stable transformants were selected with 20microg/ml G418. Our results show that expression of Eh Dia was completely inhibited in these transformants. These stable transformants could be maintained indefinitely without expression of Eh Dia. This method therefore provides an effective tool to study the phenotypic changes, which occur due to inhibition of gene expression in the absence of mutants and other microbiological manipulations in this protozoan parasite.
为了抑制溶组织内阿米巴的基因表达，我们开发了一种基于在稳定转化体中表达双链RNA干扰构建体的方法。将Eh Dia的5'端与一个插入的非特异性填充片段以头对头的方式克隆到溶组织内阿米巴表达载体pJST4中。该构建体被转化到溶组织内阿米巴HM1:IMSS滋养体中，并用20μg/ml G418筛选稳定转化体。我们的结果表明，这些转化体中Eh Dia的表达被完全抑制。这些稳定转化体可以无限期维持，而无需表达Eh Dia。因此，该方法提供了一个有效的工具来研究由于在这种原生动物寄生虫中缺乏突变体和其他微生物操作而导致的基因表达抑制所引起的表型变化。
