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首页> 外文期刊>Cell biology international. >Gene transfection and expression in a primary culture of mammary epithelial cells isolated from lactating sows.
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Gene transfection and expression in a primary culture of mammary epithelial cells isolated from lactating sows.

机译：从泌乳母猪分离的乳腺上皮细胞的原代培养中的基因转染和表达。

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摘要

Porcine mammary epithelial cells (PMECs) were isolated from lactating sow mammary glands and cultured on a matrix gel. Primary culture cells expressed significant amounts of the specific marker cytokeratin as determined by immunohistochemistry, and exhibited mammary-specific functions, such as transcription of alpha-lactalbumin, beta-casein and beta-lactoglobulin genes. They also formed mammospheres when the medium was supplemented with lactogenic hormones. The PMECs were used to study gene transfer and expression in vitro. A gene encoding enhanced green fluorescent protein (EGFP) was used as a reporter and two constructs were investigated, pEGFP-N1 (a vector constructed with a CMV promoter followed by the EGFP gene) and pGB562/GFP (a mammary gland-specific expression vector with regulatory sequences from the goat beta-casein gene linked to EGFP). The efficiency of DNA transfer into the cultured PMECs was about 20-30%. GFP expression in the pGB562/GFP-transfected PMECs was markedly stimulated by prolactin supplements in the medium. The established PMECs maintained optimal gene expression from 1 to 20 passages and appeared to provide an efficient and convenient system for assessing the expression of transgenes containing mammary gland-specific promoters.
机译：从泌乳母猪的乳腺中分离出猪乳腺上皮细胞（PMEC），并在基质凝胶上进行培养。如通过免疫组织化学测定，原代培养细胞表达大量的特异性标志物细胞角蛋白，并表现出乳腺特异性功能，例如α-乳白蛋白，β-酪蛋白和β-乳球蛋白基因的转录。当培养基中添加有乳激素时，它们也形成了乳球。 PMEC用于体外基因转移和表达的研究。使用编码增强型绿色荧光蛋白（EGFP）的基因作为报告基因，研究了两个构建体，即pEGFP-N1（由CMV启动子和EGFP基因组成的载体）和pGB562 / GFP（乳腺特异性表达载体）带有与EGFP相连的山羊β-酪蛋白基因的调控序列）。 DNA转移到培养的PMEC中的效率约为20-30％。培养基中催乳素补充剂显着刺激了pGB562 / GFP转染的PMEC中的GFP表达。已建立的PMEC在1至20代中保持了最佳的基因表达，并且似乎为评估含有乳腺特异性启动子的转基因的表达提供了一种有效而方便的系统。

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