利用TALEN技术创建RPE65基因编辑犬
Establishment of RPE65 Gene-edited Dog(Canis lupus familiaris)Using TALEN Technology
徐铮 1李振诚 2赖威仲 2许博 2钱雨玉 2陈晨 2王晶 2张勇 2冯冲 2魏彦明3
作者信息
- 1. 甘肃农业大学动物医学院,兰州 730070;甘肃省动物生殖生理及繁殖调控重点实验室,兰州 730070
- 2. 甘肃省动物生殖生理及繁殖调控重点实验室,兰州 730070
- 3. 甘肃农业大学动物医学院,兰州 730070
- 折叠
摘要
视网膜色素上皮65(retinal pigment epithelium 65,RPE65)基因在视循环中起重要作用,当RPE65基因发生突变时,视锥、视杆细胞功能丧失,严重的患者会视力丧失.为构建RPE65基因突变动物疾病模型,本研究利用转录激活因子样效应物核酸酶(transcription activator-like effector nucleases,TALENs)基因编辑技术,以比格犬(Canis lupus familiaris)为实验动物,针对RPE65基因第3号和第7号外显子构建表达载体,结合显微操作创建RPE65基因编辑犬.通过临床观察、常规眼科检查、眼底照相(fundus photography,FP)、光学相干断层扫描(optical coherence tomography,OCT)和全视野视网膜电图(electroretinography,ERG)对幼犬视功能进行表型分析检测.T7E1酶切和基因测序结果显示,最终获得2只RPE65基因编辑雄性幼犬.眼科表型分析表明,RPE65基因编辑幼犬眼底血管稀疏纤细;ERG结果显示,光感受器细胞的电位活动a波、b波振幅降低,峰时延长,提示视网膜功能受损.本研究利用TALEN基因编辑技术构建的RPE65基因编辑幼犬存在视功能障碍,可为RPE65基因突变诱导的视网膜疾病及相关研究提供参考,还为治疗手段的开发、临床药物的筛选提供良好的动物疾病模型.
Abstract
The retinal pigment epithelium 65(RPE65)gene plays an important role in the visual cycle;when the RPE65 gene is mutated,there is a loss of cone and rod cell function,and loss of vision in severe patients.In order to construct an animal disease model with RPE65 gene mutation;this study utilized transcription activator-like effector nucleases(TALEN)gene editing technology to construct expression vectors for Exon3 and Exon7 of the RPE65 gene in Beagle dogs(Canis lupus familiaris),and combined with microscopic manipulation to create RPE65 gene-edited dogs.The dogs'visual function was tested by clinical observation,routine ophthalmologic examination,fundus photography(FP),optical coherence tomography(OCT),and full-field electroretinography(ERG).T7E1 digestion and gene sequencing results showed that 2 RPE65 gene-edited male dogs were finally obtained.Ophthalmic phenotypic analysis showed that the fundus blood vessels of RPE65 gene-edited dogs were sparse and slender.The ERG results showed that the amplitude of the a-wave and b-wave potentials in photoreceptor cells decreased,and the peak time prolonged,indicating impaired retinal function.In this study,RPE65 gene-edited dogs were constructed by using TALEN gene editing technology with visual dysfunction,which may provide a reference for studies related to retinal diseases induced by mutations in the RPE65 gene,and also provide a good animal disease model for the development of therapeutic treatments and the screening of clinical drugs.
关键词
比格犬/转录激活因子样效应物核酸酶(TALEN)/视网膜色素上皮65(RPE65)/基因编辑/视网膜Key words
Beagle dog/Transcription activator-like effector nucleases(TALEN)/Retinal pigment epithelium 65(RPE65)/Gene editing/Retina引用本文复制引用
基金项目
国家自然科学基金(32060812)
甘肃省高等学校产业支撑计划(2020C-14)
甘肃省动物生殖生理及繁殖调控重点实验室经费资助项目(20JR10RA563)
出版年
2024
国家科技期刊平台
NSTL
万方数据