【摘要】：目的利用嗎啉代寡核苷酸技術(shù)建立下調(diào)斑馬魚lmna基因的技術(shù)方法。方法在斑馬魚lmna基因序列中選擇靶點(diǎn),設(shè)計(jì)針對斑馬魚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(dá)量反應(yīng)lmna基因表達(dá)量,并通過蛋白質(zhì)印跡法檢測胚胎中l(wèi)amin蛋白表達(dá)量。結(jié)果蛋白質(zhì)印跡法檢測斑馬魚體內(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)了斑馬魚胚胎lmna基因表達(dá)。結(jié)論可通過lmna-MO和重組質(zhì)粒lmna-EGFP-pCS~(2+)共注射方法下調(diào)斑馬魚lmna基因表達(dá),并通過綠色熒光蛋白表達(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)動物中心;
【基金】：國家自然科學(xué)基金項(xiàng)目資助(NO.31260284)
【分類號】：R-332

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