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首页> 外文期刊>Journal of Biotechnology >Elementary research into the transformation BmN cells mediated by the piggyBac transposon vector
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Elementary research into the transformation BmN cells mediated by the piggyBac transposon vector

机译：由piggyBac转座子载体介导的转化BmN细胞的基础研究

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摘要

To generate stable transformants of Bombyx mori silkworm BmN cells continuously expressing a target gene from a piggyBac-derived vector, BmN cells were transfected with a piggyBac vector containing a neomycin-resistance gene, green fluorescent protein gene, and human insulin-like growth factor I gene (hIGF-I) and a helper plasmid containing the piggyBac transposase sequence under the control of the B. mori actin 3 (A3) promoter. With the antibiotic G418, we selected stably transformed BmN cells expressing hIGF-I from the piggyBac-derived vector containing a neomycin-resistance gene driven by the ie-1 promoter from the B. mori nucleopolyhedrovirus. However, no stably transformed BmN cells transformed with the piggyBac element vector containing an SV40-promoter-driven neomycin-resistance gene were isolated. Determined with an enzyme-linked immunosorbent assay, the expression level of hIGF1 was about 7.8ng in 5c10e cells in which the hIGF-I gene was driven by the sericin-1 promoter, and 147.5ng in 5c10e cells in which the hIGF-I was under control of B. mori fibroin heavy chain gene (fib-H) promoter with its downstream signal peptide sequence. Analysis of the chromosomal insertion site by inverse PCR showed that the exogenous DNA was inserted into the cell genome randomly or at a TTAA target sequence, characteristic of piggyBac element transposition. These results are particularly important because piggyBac has been suggested for use in the transgenesis of silkworm cells.
机译：为了产生稳定的Bombyx mori家蚕BmN细胞转化体，使其不断从piggyBac衍生的载体表达靶基因，用​​含有新霉素抗性基因，绿色荧光蛋白基因和人胰岛素样生长因子I的piggyBac载体转染BmN细胞。基因（hIGF-1）和一个辅助质粒，该质粒在桑蚕肌动蛋白3（A3）启动子的控制下含有piggyBac转座酶序列。使用抗生素G418，我们从piggyBac衍生的载体中选择了稳定转化的表达hIGF-1的BmN细胞，该载体包含新孢子虫核多角体病毒ie-1启动子驱动的新霉素耐药基因。但是，没有分离出用含有SV40启动子驱动的新霉素抗性基因的piggyBac元件载体转化的稳定转化的BmN细胞。用酶联免疫吸附法测定，在由sericin-1启动子驱动的hIGF-I基因驱动的5c10e细胞中，hIGF1的表达水平约为7.8ng，而在hIGF-I受其抑制的5c10e细胞中，hIGF1的表达水平约为147.5ng。在家蚕纤维蛋白重链基因（fib-H）启动子的控制下，具有其下游信号肽序列。通过反向PCR分析染色体插入位点表明，外源DNA随机或以piggyBac元件转座特征的TTAA靶序列插入细胞基因组。这些结果特别重要，因为已建议将piggyBac用于家蚕细胞的转基因。

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