Wan L, Chung S K, Yang Y, Chung S S, Cao D, Ma M, Wu M, Wan Z, Chen X
Research Center of Molecular Biology, Nanhua University, Hengyang 421001, China.
Chin Med J (Engl). 2001 Oct;114(10):1078-83.
To establish a new transgenic mouse model for determining the function and role of human scavenger receptor A (SR-A) in atherosclerosis in vivo.
Human scavenger receptor minigene-driven mouse tie-1 promoter was constructed and confirmed by endonuclease digestion and sequence analysis. Transgenic mice were generated via the microinjection method. PCR and Southern blot were used to screen the positive transgenic mice. RT-PCR and immunohistochemical analysis were used to detect the level and location of human SR-AI expression in transgenic mice. The activity of human SR-AI was determined by morphologic observation of aortic endothelial cells of transgenic mice under transmission electron microscopy.
The electrophoresis assay showed the expected 4 fragments of 0.9 kb, 1.1 kb, 1.2 kb and 4.2 kb in the Sma I digest and 2 fragments of 0.8 kb and 6.7 kb in Bgl II digest of plasmids pTie-1/hSR-A. The fragment sequence of tie-1 promoter and human SR-A cDNA in plasmids pTie-1/hSR-A was correct and no ATG before the translation initiation sites of human SR-A was found by sequence analysis. 561 injected and surviving embryos with the purified human SR-A minigene were implanted into the oviducts of 19 ICR pseudopregnant mice. Among the 54 surviving pups from 13 foster mothers, 7 were identified by PCR and Southern blot analysis. The results of RT-PCR and immunohistochemical analysis showed human SR-A was specifically expressed on vascular endothelial cells of the aorta and renal artery, as well as hepatic sinusoidal endothelial cells in transgenic mice. Transmission electron microscope (TEM) of aorta of transgenic mice showed that a large number of vesicles, multivesicle bodies and swollen mitochondria filled the plasma of endothelial cells.
A transgenic mouse model with overexpression of human SR-A in endothelial cells was successfully established. The transgene was integrated and transmitted into the chromosome of transgenic mice. Tie-1 promoter controlled the transgene to express in endothelial cells in mice. Pinocytic activity of aortic endothelial cells in transgenic mice was higher than that of C57BL/6J mice. Our studies will provide a new transgenic model for investigation of atherosclerosis and functions of human SR-A.
建立一种新的转基因小鼠模型，以在体内确定人清道夫受体A（SR-A）在动脉粥样硬化中的功能和作用。
构建人清道夫受体小基因驱动的小鼠tie-1启动子，并通过核酸内切酶消化和序列分析进行确认。通过显微注射法产生转基因小鼠。采用PCR和Southern印迹法筛选阳性转基因小鼠。采用RT-PCR和免疫组织化学分析检测转基因小鼠中人SR-AI的表达水平和定位。通过透射电子显微镜对转基因小鼠主动脉内皮细胞进行形态学观察，确定人SR-AI的活性。
电泳分析显示，质粒pTie-1/hSR-A经Sma I酶切后出现预期的4条片段，大小分别为0.9 kb、1.1 kb、1.2 kb和4.2 kb，经Bgl II酶切后出现2条片段，大小分别为0.8 kb和6.7 kb。质粒pTie-1/hSR-A中tie-1启动子和人SR-A cDNA的片段序列正确，序列分析未发现人SR-A翻译起始位点之前有ATG。将561个注射了纯化人SR-A小基因且存活的胚胎植入19只ICR假孕小鼠的输卵管。在13只代孕母鼠所生的54只存活幼崽中，通过PCR和Southern印迹分析鉴定出7只为阳性。RT-PCR和免疫组织化学分析结果显示，人SR-A在转基因小鼠的主动脉和肾动脉血管内皮细胞以及肝窦内皮细胞中特异性表达。转基因小鼠主动脉的透射电子显微镜观察显示，内皮细胞质中充满大量囊泡、多泡体和肿胀的线粒体。
成功建立了内皮细胞中人SR-A过表达的转基因小鼠模型。转基因整合并传递到转基因小鼠的染色体中。Tie-1启动子控制转基因在小鼠内皮细胞中表达。转基因小鼠主动脉内皮细胞的胞饮活性高于C57BL/6J小鼠。我们的研究将为研究动脉粥样硬化和人SR-A的功能提供一种新的转基因模型。
