摘要
目的构建硒代半胱氨酸插入序列结合蛋白2(SBP2)突变型转基因小鼠模型,并分析其甲状腺表型。方法利用同源重组工程技术建立Sbp2基因突变敲入打靶载体,Southern blot筛选阳性胚胎干细胞克隆,利用显微注射法制备嵌合体小鼠,经育种后获得F3代小鼠。采用充足硒饲料和低硒饲料喂养F3代小鼠60 d,喂养30 d及60 d时采用放射性免疫法测定其甲状腺功能。结果Sbp2 R129X和R775X突变敲入打靶载体电转入胚胎干细胞后分别获得12个和23个阳性克隆,经Southern blot筛选后通过显微注射法分别获得36只Sbp2 R129X和27只Sbp2 R775X嵌合体雄鼠,育种结果显示纯合突变和复合杂合突变的F3代小鼠均在胚胎期死亡。充足硒饲料喂养下Sbp2 R129X和R775X杂合突变小鼠的甲状腺功能与野生型小鼠相比差异无统计学意义,而低硒饲料喂养下R775X杂合突变雌鼠的血清T4、雄鼠的血清rT3,以及雌鼠和雄鼠的T4/T3比值均显著增高(P<0.05)。结论成功建立了Sbp2 R129X和R775X基因突变敲入小鼠模型,低硒诱导下R775X杂合突变小鼠可模拟SBP2缺陷患者的甲状腺表型。
Objective To construct a selenocysteine insertion sequence-binding protein 2(SBP2)mutation mouse model and analyze its thyroid phenotype.Methods The Sbp2 mutant knock-in targeting vectors were established by homologous recombination engineering.Positive embryonic stem cell clones were screened by Southern blot.The mutant chimeric mice were generated by DNA microinjection,and the F3 generation was obtained after breeding.The F3 generation was fed with sufficient or low selenium food for 60 d and their thyroid function was determined by radioimmunoassay at 30 d and 60 d,respectively.Results After the Sbp2 R129X and R775X mutant knock-in targeting vectors were electroporated into embryonic stem cells,12 and 23 positive clones were obtained,respectively.After screening by Southern blot,36 Sbp2 R129X and 27 Sbp2 R775X chimeric male mice were obtained by microinjection.The breeding results showed that all homozygous mutant and compound heterozygous mutant F3 mice were embryonic lethal.Thyroid function was not significantly different between Sbp2 R129X and R775X heterozygous mutant mice and wild-type mice under the condition of sufficient selenium feeding,while the serum T4 in female,rT3 in male,and T4/T3 ratio in R775X heterozygous mutant male and female mice were significantly higher than those in wild-type mice under low selenium food(P<0.05).Conclusion Sbp2 R129X and R775X mutant mouse models are successfully established,and R775X heterozygous mutant mice under low selenium food could be used to simulate the thyroid phenotype of SBP2-deficient patients.
作者
赵洋
丁习
吕宏军
伍丽萍
邓雪阳
浮姣
ZHAO Yang;DING Xi;Lü Hongjun;WU Liping;DENG Xueyang;FU Jiao(Department of Endocrinology and Metabolism,First Affiliated Hospital of Xi’an Jiaotong University,Xi’an 710061,China;Department of Traditional Chinese Medicine Pharmacology and Traditional Chinese Medicine,China Pharmaceutical University)
出处
《山西医科大学学报》
CAS
2022年第10期1286-1293,共8页
Journal of Shanxi Medical University
基金
国家自然科学基金青年基金项目(81500597)
陕西省自然科学基础研究计划面上项目(2017JM8051)。
