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Identification of mouse lines with HA-tagged prostaglandin receptors
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- Author:
- No author available
- Journal Title:
- Acta Physiologica Sinica
- Issue:
- 4
- DOI:
- 10.13294/j.aps.2021.0050
- Key Word:
- 前列腺素受体;HA标签;转基因小鼠
Abstract： 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.
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