Ribozyme suppression of endogenous thyroid hormone receptor activity in Xenopus laevis cells.
Ribozyme suppression of endogenous thyroid hormone receptor activity in Xenopus laevis cells.
复制标题
核酶抑制非洲爪蟾细胞内源性甲状腺激素受体活性。
DOI：
10.1093/nar/gkf461
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发表时间：
2002
影响因子：
14.9
通讯作者：
Furlow,JDavid
中科院分区：
文献类型：
--
作者：
Lim,Wayland;Furlow,JDavid
非洲爪蟾是甲状腺激素（T3）调节基因表达的优秀模型。 T3 在变态过程中启动两条截然不同的途径：幼虫组织的死亡和成虫组织的生长。每种 T3 受体 (TR) 同种型 α 和 β 在变态发育中所起的作用尚不确定。 X.laevistetraploid 基因组将实验限制为过度表达、错误表达和显性失活研究。核酶通过特异性 mRNA 减少来抑制基因活性，从而提供了一种替代方案。有人认为，由于生物体的细胞内环境和体温，核酶在非洲爪蟾中不起作用。在这项研究中，我们证明锤头核酶在体外对转录的 TRβ 信息具有活性，在体内对 TRβ-荧光素酶融合蛋白具有活性。接下来，我们证明，利用来自两个 T3 响应转录因子基因的 T3 响应元件，TRβ 靶向核酶可以抑制培养的 X.laevis 细胞中 T3 诱导的报告基因转录。一种具有响应 T3 的早期表达动力学，并被认为受 TRα 调节，而另一种具有中间诱导动力学，因此可能部分受 TRβ 调节。因此，核酶是一种潜在的有价值的工具，可以克服该系统在检查非洲鲫鱼基因功能方面的局限性。
Xenopus laevisis an excellent model for thyroid hormone (T3)‐regulated gene expression. T3 initiates two drastically different pathways during metamorphosis: death of larval tissues and growth of adult tissues. The role that each T3 receptor (TR) isotype, α and β, plays in metamorphosis is uncertain. TheX.laevistetraploid genome limits experiments to overexpression, misexpression and dominant negative studies. Ribozymes offer an alternative by suppressing gene activity through specific mRNA reduction. It has been suggested that ribozymes will not work inX.laevisbecause of the organism’s intracellular environment and body temperature. In this study, we show that hammerhead ribozymes are activein vitroagainst transcribed TRβ message andin vivoagainst a TRβ–luciferase fusion protein. We next show that TRβ‐targeted ribozymes can inhibit T3‐induced transcription of a reporter gene in culturedX.laeviscells, using T3 response elements from two T3‐responsive transcription factor genes. One has early expression kinetics in response to T3 and is proposed to be TRα regulated whereas the other has intermediate induction kinetics and thus may be partially TRβ regulated. Therefore, ribozymes are a potentially valuable tool for overcoming the limitations in this system for examining gene function inX.laevis.