前列腺素受体HA标签小鼠的鉴定
陶西西, 王北, 左胜锴, 余鹰^*
天津医科大学基础医学院药理学系，天津 300070
摘要
前列腺素是一类有重要生理活性的不饱和脂肪酸衍生物，其通过结合特异性的受体发挥生理功能。前列腺素受体缺乏特异性的抗体，这极大制约着前列腺素信号直接的深入解析。本文旨在鉴定及验证“人造精子”和CRISPR-Cas9联合应用构建的氨基端(-NH2, -N)带HA标签的9种前列腺素受体小鼠。通过将向导RNA表达质粒和带标签的打靶载体质粒转入“人造精子细胞”，筛选出含HA标签的“人造精子细胞”。进一步将标签细胞注射到小鼠卵母细胞中并移植入假孕母鼠体内，获得标签小鼠。提取前列腺素受体HA标签小鼠的基因组DNA，利用PCR技术检测小鼠的基因型。分离小鼠腹腔巨噬细胞，通过Western blot方法验证带HA标签的前列腺素受体的蛋白表达情况。结果显示，前列腺素受体HA标签小鼠能扩增出特异性的DNA条带，并且巨噬细胞蛋白中可检测出特定HA蛋白条带的表达，野生型小鼠则没有相应条带。综上可知，我们成功制备了9种前列腺素受体-N-HA标签小鼠，为进一步研究前列腺素信号在体内及体外的病理生理功能提供强有力的工具。
关键词： 前列腺素受体; HA标签; 转基因小鼠
分类号：Q493.9;Q78;R339.6

Identification of mouse lines with HA-tagged prostaglandin receptors
TAO Xi-Xi, WANG Bei, ZUO Sheng-Kai, YU Ying^*
Department of Pharmacology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin 300070, China
Abstract
Prostaglandins are a class of poly-unsaturated fatty acids-derived bioactive lipids with important physiological function by binding to specific receptors. Prostaglandin receptors lack specific antibodies, which greatly impedes the research on our understanding of the signaling of prostaglandins. The aim of this study was to identify nine mouse lines with amino terminal (-NH2, -N) HA-tagged prostaglandin receptors by using the combination of artificial sperm and CRISPR-Cas9 technology. The guide RNA expression plasmid and labeled targeting vector plasmids were transferred into “artificial sperm cells”. The “artificial sperm cells” containing labeled proteins were selected and injected into mouse oocytes, and implanted into pseudopregnant mice to obtain labeled mice. The genomic DNA of the prostaglandin receptor tagged mice was extracted, and the genotypes of mice were detected by PCR method. We also isolated mouse peritoneal macrophages to verify the protein expression of HA-labeled prostaglandin receptor by Western blot. Specific DNA bands were amplified in prostaglandin receptor labeled mice, and specific HA protein bands were detected in macrophage proteins, which was not detected in wild type mice. In summary, we successfully constructed 9 mouse lines with HA-tagged prostaglandin receptors, providing a powerful tool for further study of the pathophysiological functions of prostaglandin signaling both in vivo and in vitro.


Key words: prostaglandin receptor; HA tag; transgenic mice

收稿日期：2021-02-16　　录用日期：2021-03-29
通讯作者：余鹰　　E-mail: yuying@tmu.edu.cn
DOI: 10.13294/j.aps.2021.0050
引用本文：
陶西西, 王北, 左胜锴, 余鹰. 前列腺素受体HA标签小鼠的鉴定[J]. 生理学报 2021; 73 (4): 559-570.
TAO Xi-Xi, WANG Bei, ZUO Sheng-Kai, YU Ying. Identification of mouse lines with HA-tagged prostaglandin receptors. Acta Physiol Sin 2021; 73 (4): 559-570 (in Chinese with English abstract).



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