作　　者： ; ; ; ; ;

机构地区： 南方医科大学实验动物中心与比较医学研究所

出　　处： 《中国比较医学杂志》 2019年第5期11-16,共6页

摘　　要： 目的利用CRISPR/Cas9技术敲除巴马小型猪4-羟基苯丙酮酸氧化酶(4-OH phenylpyruvate dioxygenase,4HPPD/HPD)制备高酪氨酸血症Ⅲ型巴马小型猪模型。方法分别从质粒pGL3-U6-gRNA-PGK-puromycin和pST1374-NLS-flag-linker-Cas9中通过PCR扩增sgRNA-Hpd和Cas9体外转录用模板,接着体外转录出Cas9 mRNA和sgRNA-Hpd,最后将Cas9 mRNA和sgRNA-Hpd共注射入巴马小型猪受精卵的胞质内,以制备Hpd基因敲除巴马小型猪。结果胞质内共注射后,共移植20枚受精卵至2只代孕母猪。获得子代Hpd基因敲除巴马小型猪共4只。结论本研究成功制备了高酪氨酸血症Ⅲ型巴马小型猪模型,将为研究酪氨酸代谢通路提供更好的模型。 Objective To prepare a Bama minipig model of hereditary tyrosinemia type Ⅲ, the hydroxyphenylpyruvate dioxygenase (Hpd) gene was chosen to be edited by the CRISPR/Cas9 technology. Methods Templates (used as in vitro transcripts for Cas9 mRNA and sgRNA) were amplified from pST1374-NLS-flag-linker-Cas9 and pGL3-U6-gRNA-PGK-puromycin by PCR, respectively, and subsequently transcribed in vitro into Cas9 mRNA and sgRNA-Hpd. Finally, cas9 mRNA and sgRNA-Hpd were co-injected into the cytoplasm of single cell embryos to generate an Hpd-modified Bama minipig. Results Twenty embryos of Bama minipigs co-injected with cas9 mRNA and sgRNA-Hpd were transplanted into two pseudopregnant mothers. We obtained four offsprings with a modified Hpd gene. Conclusions In this study, we have successfully generated Hpd knockout Bama minipigs with hereditary tyrosinemia type Ⅲ using the CRISPR/ Cas9 technique, which will be a valuable model for research of the tyrosine metabolic pathway.

关 键 词： 羟基苯丙酮酸氧化酶 高酪氨酸血症 型 巴马小型猪

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