【摘要】：目的利用嗎啉代寡核苷酸技術建立下調斑馬魚lmna基因的技術方法。方法在斑馬魚lmna基因序列中選擇靶點,設計針對斑馬魚lmna基因的嗎啉代寡核苷酸序列(lmna-MO),構建能特異指示lmna基因表達的lmna-EGFP-pCS~(2+)重組質粒,并通過顯微注射方式將二者共注射入斑馬魚胚胎中,通過觀察胚胎中綠色熒光表達量反應lmna基因表達量,并通過蛋白質印跡法檢測胚胎中l(wèi)amin蛋白表達量。結果蛋白質印跡法檢測斑馬魚體內lamin蛋白的表達,分別有大小為69 KD和62 KD兩種蛋白表達。設計并構建了lmna-MO和重組質粒lmnaEGFP-pCS~(2+),單獨注射lmna-EGFP-pCS~(2+)質粒后觀察到從6 hpf到96 hpf胚胎均有綠色熒光蛋白表達;二者共注射后觀察到,與對照組相比,實驗組從6 hpf至30 hpf胚胎中綠色熒光蛋白表達量均不同程度下降或消失;蛋白質印跡實驗結果顯示實驗組胚胎內lamin蛋白表達量明顯下降。表明已成功下調了斑馬魚胚胎lmna基因表達。結論可通過lmna-MO和重組質粒lmna-EGFP-pCS~(2+)共注射方法下調斑馬魚lmna基因表達,并通過綠色熒光蛋白表達量反映下調效果。該方法可為深入研究人核纖層病提供良好的動物模型。
[Abstract]:Objective to establish a method for down-regulating lmna gene of zebrafish by morpholine oligonucleotide technique. Methods the morpholine oligonucleotide sequence (lmna-MO) targeting the zebrafish lmna gene was designed to construct the recombinant plasmid lmna-EGFP-pCS~ (2), which could specifically indicate the expression of the lmna gene in zebrafish lmna gene sequence, and designed the morpholine oligonucleotide sequence (lmna-MO) targeting the zebrafish lmna gene. Both of them were co-injected into zebrafish embryos by microinjection. The expression of lmna gene was observed by observing the green fluorescence expression in embryos, and the expression of lamin protein in embryos was detected by Western blotting. Results the expression of lamin protein in zebrafish was detected by Western blot. The expression of two proteins was 69 KD and 62 KD, respectively. Lmna-MO and recombinant plasmid lmnaEGFP-pCS~ (2) were designed and constructed. Green fluorescent protein expression was observed from 6 hpf to 96 hpf embryos after single injection of lmna-EGFP-pCS~ (2) plasmid. Compared with the control group, the expression of green fluorescent protein (GFP) decreased or disappeared in 6 hpf to 30 hpf embryos in the experimental group, and the expression of lamin protein in the embryos of the experimental group was significantly decreased as compared with the control group. The results showed that the expression of lmna gene in zebrafish embryos had been down-regulated successfully. Conclusion the expression of lmna gene in zebrafish can be down-regulated by co-injection of lmna-MO and recombinant plasmid lmna-EGFP-pCS~ (2), and the down-regulation effect can be reflected by the expression of green fluorescent protein. This method can provide a good animal model for further study of human nuclear lamellar disease.
【作者單位】： 貴州醫(yī)科大學免疫學教研室;貴州醫(yī)科大學組織工程與干細胞實驗中心;貴州醫(yī)科大學附屬醫(yī)院兒科學教研室;貴州醫(yī)科大學實驗動物中心;
【基金】：國家自然科學基金項目資助(NO.31260284)
【分類號】：R-332

【相似文獻】

相關期刊論文 前10條

1 薛宏亮;張乃元;袁占鵬;;斑馬魚在神經(jīng)毒理學研究中的應用[J];公共衛(wèi)生與預防醫(yī)學;2014年02期

2 ;學科前瞻[J];當代醫(yī)學;2003年12期

3 張立鳳;鐘濤;桂永浩;;外源性視黃酸對斑馬魚心血管系統(tǒng)發(fā)育的影響[J];中國實驗動物學報;2006年02期

4 楊寒朔;湯明海;鄧洪新;楊金亮;;納升超高效液相色譜-飛行時間質譜檢測斑馬魚胚胎中小分子多肽的初步研究[J];四川大學學報(醫(yī)學版);2007年06期

5 徐娟;吳南翔;;斑馬魚在環(huán)境內分泌干擾物篩選和檢測中的應用[J];國外醫(yī)學(衛(wèi)生學分冊);2009年04期

6 胡瑋;程露陽;宋韞韜;夏鴻飛;孫大光;李鵬;李丹;陸彩玲;馬旭;;硫代硫酸鈉干擾斑馬魚胚胎發(fā)育并致畸[J];中國生物化學與分子生物學報;2009年09期

7 何秋霞;劉可春;楚杰;王思鋒;韓利文;王希敏;;斑馬魚作為模式生物在心血管疾病研究中的應用[J];生命的化學;2009年05期

8 孫淑娜;桂永浩;蔣t，

本文編號：2432094