Optimized transfection of mRNA transcribed from a d(A/T)100 tail-containing vector
Optimized transfection of mRNA transcribed from a d(A/T)100 tail-containing vector
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DOI：
10.1016/j.bbrc.2005.03.067
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发表时间：
2005-05-13
影响因子：
3.1
通讯作者：
Katz, MS
中科院分区：
文献类型：
--
作者：
Elango, N;Elango, S;Katz, MS
采用mRNA转染的研究目前受到缺乏用于产生含有长聚（A）尾的mRNA的转录载体和用于有效mRNA转染的公开方法的限制。我们构建了一个含有萤火虫荧光素酶基因的转录载体（pBS-FLuc-A100），以产生带有A（100）尾的荧光素酶mRNA，随后没有异源序列。pBS-FLuc-A100在XL 1-Blue中增殖，其中质粒比其它细菌菌株更稳定。使用TransMessenger转染试剂（Qiagen）和酵母tRNA作为载体确定最佳mRNA转染条件。萤火虫荧光素酶的表达，在转染后约12小时达到峰值，检测到低至5 ng的mRNA，并与mRNA量高达100 ng的线性。当用含有不同长度的poly（A）尾的荧光素酶mRNA转染细胞时，荧光素酶表达随着poly（A）尾长度成比例地增加，直到60 A残基，然后下降。通过该方法有效地转染了来自猴、小鼠和大鼠的细胞系。与在其5 ' UTR中含有铁应答元件的细胞铁蛋白重链mRNA类似，含有铁蛋白mRNA的5 ' UTR的转染的荧光素酶mRNA的翻译是铁依赖性的。我们的研究结果表明，poly（A）载体和转录方法描述将是有用的研究基因表达的调控在mRNA水平上的UTR。(c)2005年爱思唯尔公司All rights reserved.
Studies employing mRNA transfection are currently limited by a lack of transcription vectors for generating a long poly(A) tail-containing mRNA and published methods for efficient mRNA transfection. We have constructed a transcription vector containing firefly luciferase gene (pBS-FLuc-A100) to generate luciferase mRNA with A(100) tail followed by no heterologous sequence. The pBS-FLuc-A100 was propagated in XL1-Blue, in which the plasmid was more stable than in other bacterial strains. Optimal mRNA transfection conditions were determined using TransMessenger Transfection Reagent (Qiagen) and yeast tRNA as a carrier. Firefly luciferase expression, which peaked at about 12 h post-transfection, was detected with as little as 5 ng mRNA and was linear with mRNA amount up to 100 ng. When cells were transfected with luciferase mRNA containing different lengths of poly(A) tail, luciferase expression increased proportionally with poly(A) tail length up to 60A residues and then declined. Cell lines from monkey, mouse, and rat were transfected efficiently by this method. Like cellular ferritin heavy chain mRNA, which contains an iron response element in its 5 ' UTR, translation of transfected luciferase mRNA containing the 5 ' UTR of ferritin mRNA was iron-dependent. Our results demonstrate that the poly(A) vector and the transcription method described will be useful to study the regulation of gene expression at the mRNA level by UTRs. (c) 2005 Elsevier Inc. All rights reserved.