McGrew Michael J, Sherman Adrian, Lillico Simon G, Taylor Lorna, Sang Helen
The Roslin Institute and Royal Dick School of Veterinary Studies, University of Edinburgh, Roslin, Midlothian, UK.
BMC Dev Biol. 2010 Feb 25;10:26. doi: 10.1186/1471-213X-10-26.
Regulatory elements that control expression of specific genes during development have been shown in many cases to contain functionally-conserved modules that can be transferred between species and direct gene expression in a comparable developmental pattern. An example of such a module has been identified at the rat myosin light chain (MLC) 1/3 locus, which has been well characterised in transgenic mouse studies. This locus contains two promoters encoding two alternatively spliced isoforms of alkali myosin light chain. These promoters are differentially regulated during development through the activity of two enhancer elements. The MLC3 promoter alone has been shown to confer expression of a reporter gene in skeletal and cardiac muscle in transgenic mice and the addition of the downstream MLC enhancer increased expression levels in skeletal muscle. We asked whether this regulatory module, sufficient for striated muscle gene expression in the mouse, would drive expression in similar domains in the chicken.
We have observed that a conserved downstream MLC enhancer is present in the chicken MLC locus. We found that the rat MLC1/3 regulatory elements were transcriptionally active in chick skeletal muscle primary cultures. We observed that a single copy lentiviral insert containing this regulatory cassette was able to drive expression of a lacZ reporter gene in the fast-fibres of skeletal muscle in chicken in three independent transgenic chicken lines in a pattern similar to the endogenous MLC locus. Reporter gene expression in cardiac muscle tissues was not observed for any of these lines.
From these results we conclude that skeletal expression from this regulatory module is conserved in a genomic context between rodents and chickens. This transgenic module will be useful in future investigations of muscle development in avian species.
在许多情况下，已表明在发育过程中控制特定基因表达的调控元件包含功能保守的模块，这些模块可在物种间转移并以类似的发育模式指导基因表达。在大鼠肌球蛋白轻链（MLC）1/3基因座处已鉴定出这样一个模块的实例，该基因座在转基因小鼠研究中已得到充分表征。该基因座包含两个启动子，它们编码碱性肌球蛋白轻链的两种可变剪接异构体。这些启动子在发育过程中通过两个增强子元件的活性受到差异调节。已表明单独的MLC3启动子可在转基因小鼠的骨骼肌和心肌中赋予报告基因表达，并且添加下游MLC增强子可提高骨骼肌中的表达水平。我们询问这个足以在小鼠中驱动横纹肌基因表达的调控模块是否会在鸡的类似区域驱动表达。
我们观察到鸡MLC基因座中存在一个保守的下游MLC增强子。我们发现大鼠MLC1/3调控元件在鸡骨骼肌原代培养物中具有转录活性。我们观察到，包含该调控盒的单拷贝慢病毒插入片段能够在三个独立的转基因鸡系中以与内源性MLC基因座相似的模式驱动鸡骨骼肌快肌纤维中lacZ报告基因的表达。在这些鸡系中均未观察到心肌组织中的报告基因表达。
从这些结果我们得出结论，该调控模块在啮齿动物和鸡之间的基因组背景下骨骼肌表达是保守的。这个转基因模块将有助于未来对禽类肌肉发育的研究。
