Hpd基因修饰制备高酪氨酸血症Ⅲ型巴马小型猪模型
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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.
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.
引文
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[4] Yang H,Wang H,Jaenisch R.Generating genetically modified mice using CRISPR/Cas-mediated genome engineering [J].Nat Protoc,2014,9(8):1956-1968.
[5] Shao Y,Guan Y,Wang L,et al.CRISPR/Cas-mediated genome editing in the rat via direct injection of one-cell embryos [J].Nat Protoc,2014,9(10):2493-2512.
[6] 田国力,许洪平,朱伟明,等.新生儿苯丙酮尿症两种实验室筛查方法的质量评价 [J].检验医学,2007,22(2):171-173.
[7] Endo F,Kitano A,Uehara I,Nagata N,et al.Four-hydroxyphenylpyruvic acid oxidase deficiency with normal fumarylacetoacetase:a new variant form of hereditary hypertyrosinemia [J].Pediatr Res,1983,17:92-96
[8] Russo PA,Mitchell GA,Tanguay RM.Tyrosinemia:a review [J].Pediatr Dev Pathol,2001,4:212-221.
[2] Buckthal DJ,Roche PA,Moorehead TJ,et al.4-Hydroxyphenylpyruvate dioxygenase from pig liver [J].Methods Enzymol,1987,142:132-138
[3] Cerone R,Holme E,Schiaffino MC,et al.Tyrosinemia type III:diagnosis and ten year follow up [J].Acta Paediatr,1997,86:1013-1015
[4] Yang H,Wang H,Jaenisch R.Generating genetically modified mice using CRISPR/Cas-mediated genome engineering [J].Nat Protoc,2014,9(8):1956-1968.
[5] Shao Y,Guan Y,Wang L,et al.CRISPR/Cas-mediated genome editing in the rat via direct injection of one-cell embryos [J].Nat Protoc,2014,9(10):2493-2512.
[6] 田国力,许洪平,朱伟明,等.新生儿苯丙酮尿症两种实验室筛查方法的质量评价 [J].检验医学,2007,22(2):171-173.
[7] Endo F,Kitano A,Uehara I,Nagata N,et al.Four-hydroxyphenylpyruvic acid oxidase deficiency with normal fumarylacetoacetase:a new variant form of hereditary hypertyrosinemia [J].Pediatr Res,1983,17:92-96
[8] Russo PA,Mitchell GA,Tanguay RM.Tyrosinemia:a review [J].Pediatr Dev Pathol,2001,4:212-221.