【摘要】：目的利用嗎啉代寡核苷酸技術(shù)建立下調(diào)斑馬魚(yú)lmna基因的技術(shù)方法。方法在斑馬魚(yú)lmna基因序列中選擇靶點(diǎn),設(shè)計(jì)針對(duì)斑馬魚(yú)lmna基因的嗎啉代寡核苷酸序列(lmna-MO),構(gòu)建能特異指示lmna基因表達(dá)的lmna-EGFP-pCS~(2+)重組質(zhì)粒,并通過(guò)顯微注射方式將二者共注射入斑馬魚(yú)胚胎中,通過(guò)觀察胚胎中綠色熒光表達(dá)量反應(yīng)lmna基因表達(dá)量,并通過(guò)蛋白質(zhì)印跡法檢測(cè)胚胎中l(wèi)amin蛋白表達(dá)量。結(jié)果蛋白質(zhì)印跡法檢測(cè)斑馬魚(yú)體內(nèi)lamin蛋白的表達(dá),分別有大小為69 KD和62 KD兩種蛋白表達(dá)。設(shè)計(jì)并構(gòu)建了lmna-MO和重組質(zhì)粒lmnaEGFP-pCS~(2+),單獨(dú)注射lmna-EGFP-pCS~(2+)質(zhì)粒后觀察到從6 hpf到96 hpf胚胎均有綠色熒光蛋白表達(dá);二者共注射后觀察到,與對(duì)照組相比,實(shí)驗(yàn)組從6 hpf至30 hpf胚胎中綠色熒光蛋白表達(dá)量均不同程度下降或消失;蛋白質(zhì)印跡實(shí)驗(yàn)結(jié)果顯示實(shí)驗(yàn)組胚胎內(nèi)lamin蛋白表達(dá)量明顯下降。表明已成功下調(diào)了斑馬魚(yú)胚胎lmna基因表達(dá)。結(jié)論可通過(guò)lmna-MO和重組質(zhì)粒lmna-EGFP-pCS~(2+)共注射方法下調(diào)斑馬魚(yú)lmna基因表達(dá),并通過(guò)綠色熒光蛋白表達(dá)量反映下調(diào)效果。該方法可為深入研究人核纖層病提供良好的動(dòng)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(xué)免疫學(xué)教研室;貴州醫(yī)科大學(xué)組織工程與干細(xì)胞實(shí)驗(yàn)中心;貴州醫(yī)科大學(xué)附屬醫(yī)院兒科學(xué)教研室;貴州醫(yī)科大學(xué)實(shí)驗(yàn)動(dòng)物中心;
【基金】：國(guó)家自然科學(xué)基金項(xiàng)目資助(NO.31260284)
【分類(lèi)號(hào)】：R-332

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

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

2 ;學(xué)科前瞻[J];當(dāng)代醫(yī)學(xué);2003年12期

3 張立鳳;鐘濤;桂永浩;;外源性視黃酸對(duì)斑馬魚(yú)心血管系統(tǒng)發(fā)育的影響[J];中國(guó)實(shí)驗(yàn)動(dòng)物學(xué)報(bào);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(shí)間質(zhì)譜檢測(cè)斑馬魚(yú)胚胎中小分子多肽的初步研究[J];四川大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2007年06期

5 徐娟;吳南翔;;斑馬魚(yú)在環(huán)境內(nèi)分泌干擾物篩選和檢測(cè)中的應(yīng)用[J];國(guó)外醫(yī)學(xué)(衛(wèi)生學(xué)分冊(cè));2009年04期

6 胡瑋;程露陽(yáng);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(yú)胚胎發(fā)育并致畸[J];中國(guó)生物化學(xué)與分子生物學(xué)報(bào);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(yú)作為模式生物在心血管疾病研究中的應(yīng)用[J];生命的化學(xué);2009年05期

8 孫淑娜;桂永浩;蔣t，

本文編號(hào)：2432094