Efficient expression of transgenes in adult zebrafish by electroporation.
Efficient expression of transgenes in adult zebrafish by electroporation.
复制标题
通过电穿孔在成年斑马鱼中转基因的有效表达。
DOI：
10.1186/1472-6750-5-29
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发表时间：
2005-10-13
影响因子：
3.5
通讯作者：
Rao, NM
中科院分区：
文献类型：
--
作者：
Rambabu, KM;Rao, SHN;Rao, NM
通过注射裸DNA在肌肉中表达转基因被广泛实践。证明在注射部位施加电脉冲可改善肌肉组织中的转基因表达。斑马鱼是研究脊椎动物发育生物学的珍贵模型。在这项研究中，我们研究了电穿孔对3-6月龄成年斑马鱼转基因表达的影响。对脉冲数、电压和质粒DNA量等电穿孔参数进行优化，结果表明，当脉冲数为40 V·cm-1，质粒DNA量为15 μg/尾，电穿孔6次时，荧光素酶的表达量是对照组的10倍。在印度鲤鱼（野Labeo rohita）中也观察到类似的转基因表达增强。为了建立成年斑马鱼作为瞬时转染系统的实用性，在电穿孔后在A2细胞和成年斑马鱼中比较启动子的强度。发现启动子的相对强度在细胞系和成年斑马鱼中是相似的。还通过荧光显微镜研究了电穿孔后组织中的GFP荧光。DNA注射后的电穿孔使成年斑马鱼的基因表达增强10倍。提出了镊子型电极电穿孔转染成年斑马鱼的最佳参数。电穿孔后增强的报告基因表达允许在斑马鱼体内比较启动子的强度。
Expression of transgenes in muscle by injection of naked DNA is widely practiced. Application of electrical pulses at the site of injection was demonstrated to improve transgene expression in muscle tissue. Zebrafish is a precious model to investigate developmental biology in vertebrates. In this study we investigated the effect of electroporation on expression of transgenes in 3–6 month old adult zebrafish. Electroporation parameters such as number of pulses, voltage and amount of plasmid DNA were optimized and it was found that 6 pulses of 40 V·cm-1 at 15 μg of plasmid DNA per fish increased the luciferase expression 10-fold compared to controls. Similar enhancement in transgene expression was also observed in Indian carp (Labeo rohita). To establish the utility of adult zebrafish as a system for transient transfections, the strength of the promoters was compared in A2 cells and adult zebrafish after electroporation. The relative strengths of the promoters were found to be similar in cell lines and in adult zebrafish. GFP fluorescence in tissues after electroporation was also studied by fluorescence microscopy. Electroporation after DNA injection enhances gene expression 10-fold in adult zebrafish. Electroporation parameters for optimum transfection of adult zebrafish with tweezer type electrode were presented. Enhanced reporter gene expression upon electroporation allowed comparison of strengths of the promoters in vivo in zebrafish.
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影响因子：
1.5
作者：
Tawk, M;Tuil, D;Paulin, D
通讯作者：
Paulin, D
DOI：
10.1023/a:1022927822244
发表时间：
2002-11-01
期刊：
Somatic Cell and Molecular Genetics
影响因子：
--
作者：
Fattori, Elena;La Monica, Nicola;Toniatti, Carlo
通讯作者：
Toniatti, Carlo
影响因子：
2.9
作者：
Ogura, T
通讯作者：
Ogura, T
影响因子：
3
作者：
Sudha, PM;Low, S;Gong, ZY
通讯作者：
Gong, ZY
影响因子：
3.1
作者：
Xu, YF;He, JY;Gong, ZY
通讯作者：
Gong, ZY