基于CRISPR/Cas9构建小鼠UOX基因敲除模型(1)
[摘要] 目的 通过 CRISPR/Cas9获得UOX基因敲除的小鼠纯合品系，为建立高尿酸血小鼠动物模型奠定基础。方法根据小鼠 UOX 基因第三外显子前后两个位点设计双sgRNA，通过PCR、体外转录和纯化获得sgRNA和Cas9 mRNA。将sgRNA和Cas9 mRNA显微注射进小鼠原核胚后，体外培养至二细胞胚阶段，进行胚胎移植至代孕母鼠。F0代小鼠出生后，提取其DNA进行电泳分析和测序分析。F0代交配繁殖至F2代获得UOX缺失的小鼠纯合品系。 结果 显微注射sgRNA和Cas mRNA至原核胚，成功获得50枚囊胚。移植后生育17只幼鼠。其中，有 8只小鼠UOX基因缺失突变，突变率为47.06 %。成功构建了UOX缺失的小鼠纯合品系。 结论 利用 CRISPR/Cas9，成功获得 UOX缺失的小鼠胚胎和纯合品系，为CRISPR/Cas9获得高尿酸血的小鼠动物模型奠定基础。
[關键词] CRISPR/Cas9;UOX;基因编辑;高尿酸;动物模型
[中图分类号] R4 [文献标识码] A [文章编号] 1674-0742(2020)03(a)-0032-04
Construction of Mouse UOX Gene Knockout Model Based on CRISPR / Cas9
ZHANG Ru-jun1， XIA Hai-lin1， ZHU Yun2， HUANG Jing3， MENG Yu-han2
1.Changzhou Cavens Experimental Animal Co.， Ltd.， Changzhou， Jiangsu Province， 213104 China;2.Jiangsu Kebiao Medical Testing Co.， Ltd.， Changzhou，Jiangsu Province， 213461 China;3.Baige Gene Technology (Jiangsu) Co.， Ltd.，Chang zhou， Jiangsu Province， 213000 China
[Abstract] Objective To obtain homozygous strains of UOX knockout mice by CRISPR / Cas9， and to lay the foundation for establishing an animal model of hyperuricemia mice. Methods Double sgRNA was designed based on two sites before and after the third exon of mouse UOX gene， and sgRNA and Cas9 mRNA were obtained by PCR， in vitro transcription and purification. After microinjection of sgRNA and Cas9 mRNA into mouse prokaryotic embryos， they were cultured in vitro to the two-cell embryo stage， and embryos were transferred to surrogate mother rats. After F0 mice were born， their DNA was extracted for electrophoretic analysis and sequencing analysis. FOX generations were bred to F2 generations to obtain UOX-deficient mouse homozygous lines. Results Microinjection of sgRNA and Cas mRNA into prokaryotic embryos successfully obtained 50 blastocysts. After transplantation， 17 young rats were born. Among them， 8 mice had UOX gene deletion mutations， and the mutation rate was 47.06%. A homozygous mouse line lacking UOX was successfully constructed. Conclusion UCRISPR-deficient mouse embryos and homozygous strains were successfully obtained using CRISPR / Cas9， laying a foundation for CRISPR / Cas9 to obtain hyperuricemia mouse animal models.
[Key words] CRISPR / Cas9; UOX; Gene editing; Hyperuricemia; Animal model
高尿酸血症(hyperuricemia)是由嘌呤代谢紊乱，尿酸排泄障碍引起的血尿酸异常为临床表现的异质性疾病。尿酸病理性升高有5%～12%的风险导致尿酸结晶累积并损伤关节[1]，引起痛风、急性及慢性关节炎等。并且，高尿酸血症易引发并发症，如高血压、高血脂、2型糖尿病、冠心病。临床上常规的高尿酸血症及痛风治疗手段为口服降尿酸药物[2]，目前尚未有根治痛风的药物，新型降尿酸药物开发需要利用高尿酸动物模型进行降尿酸药物药效及药理筛选。故建立稳定性好，更接近患者发病特征的高尿酸动物模型，能提高新型降尿酸药物开发效率及成药性，优化痛风治疗格局。近年来，CRISPR/Cas9基因编辑技术凭借其简便性、特异性和高效性，在疾病动物模型构建领域应用日趋广泛和深入。该研究利用CRISPR/Cas9，敲除小鼠基因组内尿酸氧化酶(Urate Oxidase，UOX)基因，以诱导小鼠尿酸分解障碍，为建立高尿酸血小鼠动物模型奠定基础，现报道如下。, 百拇医药(张茹君 夏海林 朱赟 黄晶 孟雨菡)
[關键词] CRISPR/Cas9;UOX;基因编辑;高尿酸;动物模型
[中图分类号] R4 [文献标识码] A [文章编号] 1674-0742(2020)03(a)-0032-04
Construction of Mouse UOX Gene Knockout Model Based on CRISPR / Cas9
ZHANG Ru-jun1， XIA Hai-lin1， ZHU Yun2， HUANG Jing3， MENG Yu-han2
1.Changzhou Cavens Experimental Animal Co.， Ltd.， Changzhou， Jiangsu Province， 213104 China;2.Jiangsu Kebiao Medical Testing Co.， Ltd.， Changzhou，Jiangsu Province， 213461 China;3.Baige Gene Technology (Jiangsu) Co.， Ltd.，Chang zhou， Jiangsu Province， 213000 China
[Abstract] Objective To obtain homozygous strains of UOX knockout mice by CRISPR / Cas9， and to lay the foundation for establishing an animal model of hyperuricemia mice. Methods Double sgRNA was designed based on two sites before and after the third exon of mouse UOX gene， and sgRNA and Cas9 mRNA were obtained by PCR， in vitro transcription and purification. After microinjection of sgRNA and Cas9 mRNA into mouse prokaryotic embryos， they were cultured in vitro to the two-cell embryo stage， and embryos were transferred to surrogate mother rats. After F0 mice were born， their DNA was extracted for electrophoretic analysis and sequencing analysis. FOX generations were bred to F2 generations to obtain UOX-deficient mouse homozygous lines. Results Microinjection of sgRNA and Cas mRNA into prokaryotic embryos successfully obtained 50 blastocysts. After transplantation， 17 young rats were born. Among them， 8 mice had UOX gene deletion mutations， and the mutation rate was 47.06%. A homozygous mouse line lacking UOX was successfully constructed. Conclusion UCRISPR-deficient mouse embryos and homozygous strains were successfully obtained using CRISPR / Cas9， laying a foundation for CRISPR / Cas9 to obtain hyperuricemia mouse animal models.
[Key words] CRISPR / Cas9; UOX; Gene editing; Hyperuricemia; Animal model
高尿酸血症(hyperuricemia)是由嘌呤代谢紊乱，尿酸排泄障碍引起的血尿酸异常为临床表现的异质性疾病。尿酸病理性升高有5%～12%的风险导致尿酸结晶累积并损伤关节[1]，引起痛风、急性及慢性关节炎等。并且，高尿酸血症易引发并发症，如高血压、高血脂、2型糖尿病、冠心病。临床上常规的高尿酸血症及痛风治疗手段为口服降尿酸药物[2]，目前尚未有根治痛风的药物，新型降尿酸药物开发需要利用高尿酸动物模型进行降尿酸药物药效及药理筛选。故建立稳定性好，更接近患者发病特征的高尿酸动物模型，能提高新型降尿酸药物开发效率及成药性，优化痛风治疗格局。近年来，CRISPR/Cas9基因编辑技术凭借其简便性、特异性和高效性，在疾病动物模型构建领域应用日趋广泛和深入。该研究利用CRISPR/Cas9，敲除小鼠基因组内尿酸氧化酶(Urate Oxidase，UOX)基因，以诱导小鼠尿酸分解障碍，为建立高尿酸血小鼠动物模型奠定基础，现报道如下。, 百拇医药(张茹君 夏海林 朱赟 黄晶 孟雨菡)