Engineering Research Centre for Transgenic Animal Pharmaceutics in Jiangsu Province, College of Veterinary Medicine, Yangzhou University, No. 12 Wenhui Rd., Yangzhou, Jiangsu, PR China.
Mol Reprod Dev. 2012 Aug;79(8):573-85. doi: 10.1002/mrd.22063. Epub 2012 Jul 9.
It is very important to develop an effective, specific, and robust expression cassette that ensures a high level of expression in the mammary glands. In this study, we designed and constructed a series of mammary gland-specific vectors containing a complex hybrid promoter/enhancer by utilizing promoter sequences from milk proteins (i.e., goat β-casein, bovine αs1-casein, or goat β-lactoglobulin) and cytomegalovirus enhancer sequences; vectors containing a single milk protein promoter served as controls. Chicken β-globin insulator sequences were also included in some of these vectors. The expression of constructs was analyzed through the generation of transgenic mice. Enzyme-linked immunosorbent assay (ELISA) analysis revealed that the hybrid promoter/enhancer could drive the expression of recombinant human lactoferrin (rhLF) cDNA at high levels (1.17-8.10 mg/ml) in the milk of transgenic mice, whereas control promoters achieved a very low rhLF expression (7-40 ng/ml). Moreover, the expression of rhLF was not detected in the serum or saliva of any transgenic animal. This result shows that all constructs, driven by the hybrid promoter/enhancer, had high mammary gland-specific expression pattern. Together, our results suggest that the use of a hybrid promoter/enhancer is a valuable alternative approach for increasing mammary-specific expression of recombinant hLF in a transgenic mouse model.
开发一种有效的、特异性强的、稳健的表达载体对于确保乳腺中高水平的表达非常重要。在这项研究中,我们设计并构建了一系列含有复杂的乳腺特异性启动子/增强子的载体,该启动子/增强子利用了乳蛋白(即山羊β-酪蛋白、牛αs1-酪蛋白或山羊β-乳球蛋白)的启动子序列和巨细胞病毒增强子序列;载体含有单个乳蛋白启动子作为对照。在这些载体中的一些中还包含了鸡β-球蛋白绝缘子序列。通过生成转基因小鼠来分析构建体的表达。酶联免疫吸附测定(ELISA)分析显示,杂交启动子/增强子可以在转基因小鼠的乳汁中高水平地表达重组人乳铁蛋白(rhLF)cDNA(1.17-8.10mg/ml),而对照启动子只能实现非常低的 rhLF 表达(7-40ng/ml)。此外,在任何转基因动物的血清或唾液中都没有检测到 rhLF 的表达。这一结果表明,所有由杂交启动子/增强子驱动的构建体均具有高乳腺特异性表达模式。总之,我们的结果表明,使用杂交启动子/增强子是提高转基因小鼠模型中重组 hLF 乳腺特异性表达的一种有价值的替代方法。