Li Yan, Zhou Mingqian, Zhou Hongwei, Ning Yunshan
School of Biotechnology, Southern Medical University, North1838 Guangzhou Road, Guangzhou, 510515, People's Republic of China,
Funct Integr Genomics. 2014 Mar;14(1):229-36. doi: 10.1007/s10142-013-0348-x. Epub 2013 Nov 28.
Introns can enhance gene expression levels. This effect is known as intron-mediated enhancement, which is different from that of enhancers or promoters. In our previous study, under the control of the cytomegalovirus or goat β-casein promoter, the vector containing intron V-TPOcDNA expressed the highest thrombopoietin (TPO) level, whereas the vector containing TPOgDNA expressed the lowest level. In order to verify whether intron V also improves TPO expression in the milk of transgenic mice, rat whey acidic protein promoter was used as regulatory element to construct mammary gland expression vectors including pTPOWA (containing TPOcDNA), pTPOWB (containing intron V-TPOcDNA), and pTPOWC (containing TPOgDNA). These vectors were transfected into HC-11 cells and the supernatants were analyzed at 48 h. The highest TPO level was found in pTPOWB (795 pg/mL) and the lowest level in pTPOWC (193 pg/mL). Then, corresponding vectors were microinjected into fertilized mice zygotes. Transgenic mice were identified by polymerase chain reaction and Southern blot. Enzyme-linked immunosorbent assay was performed to measure TPO levels in the milk of lactating transgenic mice. The highest and lowest TPO levels were found in transgenic mice carrying intron V-TPOcDNA (2,307 pg/mL) and in transgenic mice carrying TPOgDNA (242 pg/mL), respectively. Thus, intron V remarkably improved TPO expression in transgenic mice.
内含子可提高基因表达水平。这种效应被称为内含子介导的增强作用，它与增强子或启动子的作用不同。在我们之前的研究中，在巨细胞病毒或山羊β-酪蛋白启动子的控制下，含有内含子V-TPO cDNA的载体表达的血小板生成素（TPO）水平最高，而含有TPO gDNA的载体表达水平最低。为了验证内含子V是否也能提高转基因小鼠乳汁中TPO的表达，将大鼠乳清酸性蛋白启动子用作调控元件，构建了乳腺表达载体，包括pTPOWA（含TPO cDNA）、pTPOWB（含内含子V-TPO cDNA）和pTPOWC（含TPO gDNA）。将这些载体转染到HC-11细胞中，并在48小时后分析上清液。发现pTPOWB中的TPO水平最高（795 pg/mL），pTPOWC中的水平最低（193 pg/mL）。然后，将相应的载体显微注射到受精的小鼠受精卵中。通过聚合酶链反应和Southern印迹法鉴定转基因小鼠。进行酶联免疫吸附测定以测量泌乳转基因小鼠乳汁中的TPO水平。分别在携带内含子V-TPO cDNA的转基因小鼠（2307 pg/mL）和携带TPO gDNA的转基因小鼠（242 pg/mL）中发现了最高和最低的TPO水平。因此，内含子V显著提高了转基因小鼠中TPO的表达。
