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西农萨能羊FAS基因shRNA序列筛选及其腺病毒载体的构建
投稿时间:2009-05-27  修订日期:2009-10-09  点此下载全文
引用本文:王 伟,罗 军,赵旺生,李建华,张 晓,王龙坛.西农萨能羊FAS基因shRNA序列筛选及其腺病毒载体的构建[J].西北农业学报,2010,19(3):6~12
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作者单位
王 伟,罗 军,赵旺生,李建华,张 晓,王龙坛 (西北农林科技大学 动物科技学院陕西省农业分子生物学重点实验室陕西杨陵 712100) 
基金项目:国家高技术研究发展计划(863计划)项目(2008AA10Z135)资助。
中文摘要:山羊脂肪酸合酶 (Fatty acid synthase,FAS)是脂肪酸合成的关键酶,对乳腺短、中链脂肪酸合成起重要调控作用。设计了shRNA-5544、shRNA-5936、shRNA-6132 3条针对FAS基因不同区域的小发夹RNA(Short hairpin RNA,shRNA)及一条阴性对照序列shRNA-NC,并构建表达这4条shRNA序列的入门载体及其靶基因与红色荧光蛋白基因的融合表达载体,二者共转染HEK 293细胞进行有效序列筛选,结果显示shRNA-5544和shRNA-5936序列具有明显的干扰效果。在LR Clonase-Ⅱ重组酶作用下,分别将pENTR/CMV-GFP/U6-shRNA-5544、pENTR/CMV-GFP/U6-shRNA-5936及pENTR/CMV-GFP/U6-shRNA-NC入门载体与腺病毒骨架载体pAd/PL-DEST进行LR重组,经氨苄青霉素及氯霉素抗性筛选后成功获得3个重组腺病毒载体,ScaⅠ酶切鉴定及测序分析证实所构建的重组腺病毒载体中插入序列与设计序列一致。重组腺病毒载体经PacⅠ 酶切线性化后,利用Lipofectamine 2000转染HEK 293细胞,10~12 d后收集病毒,在HEK 293细胞中反复扩增3次后,获得高滴度的重组腺病毒,利用TCID50法测定重组腺病毒滴度分别为6×108 PFU/mL(表达shRNA-5544序列)、5×108 PFU/mL(表达shRNA-5936序列)及6×108 PFU/mL(表达shRNA-NC序列),为进一步在原代培养的山羊乳腺上皮细胞中进行FAS基因的RNA干扰研究奠定基础。
中文关键词:西农萨能羊  乳腺  脂肪酸合酶基因  shRNA  腺病毒载体
 
Screening of shRNA Sequence Target Xinong Saanen Goat FAS Gene and the Construction of Recombinant Adenovirus Vector
Abstract:Goat fatty acid synthase (FAS),a central multifunctional enzyme in charge of fatty acid synthesis in mammary gland,plays a significant role in regulation of short-and medium-chain fatty acid. This study designed three short hairpin RNA (shRNA) sequences shRNA-5544,shRNA-5936,and shRNA-6132 targeting different area of FAS gene and one negative control sequence,then constructed four entry vectors containing shRNA expression cassette as well as pDsRed1-C1 vector expressing target gene by cotransfecting HEK 293 cell. The result showed that entry vector expressing shRNA-5544 and shRNA-5936 sequences caused an obvious interference effect. Then ampicillin and chloramphenicol were successfully applied to identify three recombinant adenovirus vectors generated by LR recombination between entry vector (pENTR/CMV-GFP/U6-shRNA-5544,pENTR/CMV-GFP/U6-shRNA-5936,pENTR/CMV-GFP/U6-shRNA-NC) and backbone vector (pAd/PL-DEST). ScaⅠdigestion identification and sequencing analysis indicated that the sequence inserted into pAd/PL-DEST vector was the same with designed shRNA template. Recombinant adenovirus vector was transfected into HEK 293 Cell by Lipofectamine 2000 reagent after linearization by PacⅠdigestion. The cell lysates harvested during 10 to 12 days post transfection were employed to perform virus amplification in HEK 293 cells for three times to generate high-titer virus. The titer of the adenoviral stock was determined by TCID50 method and respectively reaches 6×108 PFU/mL(Expressing shRNA-5544 sequence),5×108 PFU/mL(Expressing shRNA-5936 sequence),and 6×108 PFU/mL(Expressing shRNA-NC sequence),respectively. The attaining of recombinant virus lays a foundation for RNA interference research of FAS gene in primary goat mammary epithelial cells.
keywords:Xinong Saanen goat  Mammary gland  Fatty acid synthase gene  shRNA  Adenovirus vector
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