靶向调控大鼠海马CNN3基因表达载体的构建及鉴定
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摘要
目的建立可调控大鼠局部脑区CNN3基因表达的技术,为进一步研究CNN3基因参与脑内病理生理过程奠定基础。方法设计并合成针对CNN3基因的全长编码序列c DNA和3条sh RNA干扰靶点序列,利用基因工程技术构建CNN3-OE和3个CNN3-sh RNA慢病毒载体,在立体定位仪引导下注入大鼠海马,采用蛋白免疫印迹技术筛选CNN3基因的最佳沉默序列,并找出重组表达和沉默慢病毒载体调控海马CNN3基因的变化规律。结果CNN3-OE和3个CNN3-sh RNA慢病毒干扰载体均构建成功,转染后8周内对海马CNN3基因水平均有一定调控作用,其中CNN3-sh RNA2载体组在转染后的8周内海马CNN3基因编码蛋白calponin-3水平均显著性下调,最高抑制率为73.26%;CNN3-OE慢病毒载体组中海马calponin-3蛋白水平具有统计学意义的升高仅在转染后第14天,上调率为93.88%。结论通过定位注射CNN3-OE和CNN3-sh RNA慢病毒载体可在体调控局部脑区CNN3基因表达,为后续的机制研究和开拓疾病防治新途径奠定基础。
Objective To establish a method focusing on regulation of CNN3 gene in the rat hippocampus and help to explore the role of CNN3 gene played in the brain physiology and pathology. Methods One c DNA sequence and three sh RNAs targeting CNN3 gene were designed and synthesized. The recombinant lentivirus-mediated expressing and three short hairpin RNA( sh RNA) vectors targeting CNN3 gene in the rats were constructed with engineering technology. All recombinant vectors were intravenously injected into rats hippocampi guided by stereotaxic apparatus. Western blot was performed to explore the best sh RNA and to study the changes of CNN3 gene in the rat hippocampus after transfection with the silence and over-expressed vectors. Results The lentivirus-mediated vector expressing CNN3-OE and three sh RNA vectors targeting CNN3 gene were successfully constructed. Within eight weeks after transfection,the vectors of CNN3-OE and three CNN3-sh RNAs changed the expression of CNN3 gene in the rat hippocampus,in particular,all the protein levelsof calponin-3 encoded by CNN3 gene were significantly down-regulated along with the time,with the highest inhibitory rate of 73. 6% in the CNN3-sh RNA2 group. Significant up-regulation of calponin-3 protein level by 93. 88%,was found only on the 14 th day after transfection. Conclusions Lentivirus-mediated vectors of CNN3-OE and CNN3-sh RNAs may regulate in vivo the CNN3 gene level in the local brain region of rats via stereotactic injection. The study lays a foundation for disease prevention and treatment in the future.
Objective To establish a method focusing on regulation of CNN3 gene in the rat hippocampus and help to explore the role of CNN3 gene played in the brain physiology and pathology. Methods One c DNA sequence and three sh RNAs targeting CNN3 gene were designed and synthesized. The recombinant lentivirus-mediated expressing and three short hairpin RNA( sh RNA) vectors targeting CNN3 gene in the rats were constructed with engineering technology. All recombinant vectors were intravenously injected into rats hippocampi guided by stereotaxic apparatus. Western blot was performed to explore the best sh RNA and to study the changes of CNN3 gene in the rat hippocampus after transfection with the silence and over-expressed vectors. Results The lentivirus-mediated vector expressing CNN3-OE and three sh RNA vectors targeting CNN3 gene were successfully constructed. Within eight weeks after transfection,the vectors of CNN3-OE and three CNN3-sh RNAs changed the expression of CNN3 gene in the rat hippocampus,in particular,all the protein levelsof calponin-3 encoded by CNN3 gene were significantly down-regulated along with the time,with the highest inhibitory rate of 73. 6% in the CNN3-sh RNA2 group. Significant up-regulation of calponin-3 protein level by 93. 88%,was found only on the 14 th day after transfection. Conclusions Lentivirus-mediated vectors of CNN3-OE and CNN3-sh RNAs may regulate in vivo the CNN3 gene level in the local brain region of rats via stereotactic injection. The study lays a foundation for disease prevention and treatment in the future.
引文
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[13]Molofsky AV,Kelley KW,Tsai HH,et al.Astrocyte-encoded positional cues maintain sensorimotor circuit integrity[J].Nature,2014,509(7499):189-194.
[14]石宏,何伟,王晓宇,等.TRPV1和ASIC3基因敲除小鼠的寿命观察[J].中国比较医学杂志,2014,24(10):78-81.