FAH基因敲除克隆小型猪的制备及繁育
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摘要
目的供体的短缺是器官移植所面临的主要问题,制备人源化器官是解决此问题的有效途径之一。缺乏延胡索酸乙酰乙酸水解酶(fumarylacetoacetate hydrolase, FAH)会引起遗传性酪氨酸血症Ⅰ型(hereditary tyrosinemia typeⅠ, HTⅠ),致使患者肝细胞凋亡,呈现出肝损伤。对猪(Sus scrofa)进行基因编辑,建立FAH基因敲除巴马小型猪,结合人干细胞将有利于人源化干细胞肝的制备。本研究在α-1,3-半乳糖基转移酶基因(α-1, 3-galactosyltransferase,GGTA1)敲除巴马小型猪的基础上,通过CRISPR/Cas9技术制备FAH基因敲除(FAH~(+/-)、FAH~(-/-))小型猪,并分析其健康与繁育状况。方法针对猪FAH基因设计单链导向RNA (single guide RNA, sgRNA)构建pX330表达载体转染巴马小型猪耳成纤维细胞(ear fibroblasts of Bama minipigs, BMEF)并进行敲除效率验证,鉴定后选择阳性细胞作为核供体,通过体细胞核移植技术制备FAH基因敲除巴马小型猪。提取仔猪的基因组DNA,经PCR测序后得到突变类型;采集5月龄FAH~(+/-)巴马小型猪血液,检测其血生化与血常规指标并观察肝组织学染色切片;当FAH~(+/-)巴马小型猪公猪性成熟后,与野生型(wild type, WT)母猪配种,以其产仔数评价繁育能力状况。结果得到了FAH~(+/-)巴马小型猪,Western blot显示FAH~(+/-)巴马小型猪的肝和肾中FAH的表达量均有所下降;FAH~(+/-)猪与野生型相比,FAH~(+/-)猪血液理化指标无显著差异;组织学切片染色发现FAH~(+/-)猪肝组织形态出现空泡样变化;与FAH~(+/-)公猪配种的野生母猪产仔数正常;FAH~(+/-)猪具有正常的健康状况和繁育能力。结论本研究制备出了FAH单基因敲除小型猪,健康状况及繁育能力正常,该单基因敲除猪为将来制备出双等位基因敲除猪与人源化肝的研究提供了良好基础。
Objective The shortage of donors is a major problem in organ transplantation, and the generation of humanized organs is an effective method to solve this problem. A lack of fumarylacetoacetate hydrolase(FAH) causes hereditary tyrosinemia type I(HTI), which leads to hepatocyte apoptosis and liver damage. Genetic editing of pigs(Sus scrofa), establishment of FAH knockout Bama minipigs, combined with human stem cells will facilitate the production of humanized stem cell liver. In this study, FAH gene knockout Bama minipigs were generated by CRISPR/Cas9 based on alpha-1,3-galactosyltransferase(GGTA1) gene knockout pigs,and their health and breeding status were analyzed. Methods Single guide RNA(sgRNAs) were designed to target the porcine FAH gene to construct a pX330 vector that was transfected into Bama minipig ear fibroblasts. The efficiency of this method was verified. After identification, positive cells were selected as nuclear donors, and FAH gene knockout Bama minipigs were generated by somatic cell nuclear transplantation technology. The genomic DNA of piglets was extracted and the mutant type was obtained by PCR sequencing. FAH~(+/-) Bama minipig blood was collected at the age of 5 months, and its blood biochemical and blood routine indexes were tested. Histological changes of livers were analyzed. When FAH~(+/-) Bama minipigs were sexually mature, they were mated with wild type(WT) sows, and their breeding ability was evaluated by litter size. Results FAH~(+/-) and FAH~(-/-) Bama minipigs were obtained. Western blotting showed that FAH~(+/-) Bama minipigs had decreased FAH expression in the liver and kidney. There were no significant differences in the physical and chemical indexes of blood from the FAH~(+/-) Bama minipigs or WT minipigs. Histological examination revealed vacuolar-like changes in the FAH~(+/-) pig liver tissues. The litter size of wild sows bred with FAH~(+/-) boars was normal. FAH~(+/-) pigs had normal health status and breeding ability. Conclusions FAH single allele knockout pigs are generated in this study, with normal health and reproductive abilities, which provides a good basis for future studies of the production of double-allele knockout pigs and humanized liver.
Objective The shortage of donors is a major problem in organ transplantation, and the generation of humanized organs is an effective method to solve this problem. A lack of fumarylacetoacetate hydrolase(FAH) causes hereditary tyrosinemia type I(HTI), which leads to hepatocyte apoptosis and liver damage. Genetic editing of pigs(Sus scrofa), establishment of FAH knockout Bama minipigs, combined with human stem cells will facilitate the production of humanized stem cell liver. In this study, FAH gene knockout Bama minipigs were generated by CRISPR/Cas9 based on alpha-1,3-galactosyltransferase(GGTA1) gene knockout pigs,and their health and breeding status were analyzed. Methods Single guide RNA(sgRNAs) were designed to target the porcine FAH gene to construct a pX330 vector that was transfected into Bama minipig ear fibroblasts. The efficiency of this method was verified. After identification, positive cells were selected as nuclear donors, and FAH gene knockout Bama minipigs were generated by somatic cell nuclear transplantation technology. The genomic DNA of piglets was extracted and the mutant type was obtained by PCR sequencing. FAH~(+/-) Bama minipig blood was collected at the age of 5 months, and its blood biochemical and blood routine indexes were tested. Histological changes of livers were analyzed. When FAH~(+/-) Bama minipigs were sexually mature, they were mated with wild type(WT) sows, and their breeding ability was evaluated by litter size. Results FAH~(+/-) and FAH~(-/-) Bama minipigs were obtained. Western blotting showed that FAH~(+/-) Bama minipigs had decreased FAH expression in the liver and kidney. There were no significant differences in the physical and chemical indexes of blood from the FAH~(+/-) Bama minipigs or WT minipigs. Histological examination revealed vacuolar-like changes in the FAH~(+/-) pig liver tissues. The litter size of wild sows bred with FAH~(+/-) boars was normal. FAH~(+/-) pigs had normal health status and breeding ability. Conclusions FAH single allele knockout pigs are generated in this study, with normal health and reproductive abilities, which provides a good basis for future studies of the production of double-allele knockout pigs and humanized liver.
引文
[1] Kvittingen EA,Rootwelt H,Van DT,et al.Hereditary tyrosinemia typeⅠ:lack of correlation between clinical findings and amount of immunoreactivve fumarylacetoacetase protein[J].Pediatr Res,1992,31(1):43-46.
[2] Jorquera R,Tanguay RM.The mutagenicity of the tyrosine metabolite,fumarylacetoacetate,is enhanced by glutathione depletion [J].Biochem Biophys Res Commun,1997,232(1):42-48.
[3] Orejuela D,Jorquera R,Bergeron A,et al.Hepatic stress in hereditary tyrosinemia type Ⅰ (HTⅠ) activates the AKT survival pathway in the fah-/- knockout mice model [J].J Hepatol,2008,48(2):308-317.
[4] Qi Z,Wang X,Wei H,et al.Infiltrating neutrophils aggravate metabolic liver failure in fah deficient mice [J].Liver Int,2015,35(3):774-785.
[5] Lindblad B,Lindstedt S,Steen G.On the enzymic defect in hereditary tyrosinemia [J].Proc Natl Acad Sci U S A,1977,74(10):4641-4645.
[6] Hancock ML,Meyer RC,Mistry M,et al.Insulin receptor associates with promoters genome-wide and regulates gene expression [J].Cell,2019,177(3):722-736.e22.
[7] Masurel-Paulet A,Poggi-Bach J,Rolland MO,et al.NTBC treatment in tyrosinaemia type Ⅰ:long-term outcome in French patients [J].J Inherit Metab Dis,2008,31(1):81-87.
[8] Ellis MK,Whitfield AC,Gowans LA,et al.Inhibition of 4-hydroxyphenylpyruvate dioxygenase by 2-(2-nitro-4-trifluoromethylbenzoyl)-cyclohexane-1,3-dione and 2-(2-chloro-4-methanesulfonylbenzoyl)-cyclohexane-1,3-dione[J].Toxicol Appl Pharmacol,1995,133(1):12-19
[9] Holme E,Lindstedt S.Tyrosinaemia type Ⅰ and NTBC (2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione) [J].J Inherit Metab Dis,1998,21(5):507-517.
[10] Molchanov S,Gryffkeller A.Inhibition of 4-hydroxyphenylpyruvate dioxygenase by 2-[2-nitro-4-(trifluoromethyl) benzoyl]-1,3-cyclohexanedione[J].Acta Biochim Pol,2009,56(3):447-454.
[11] 陈钟,陈瑞新,丁义涛,等.新型生物人工肝系统治疗急性肝衰竭犬后猪内源性逆转录病毒传播的实验研究[J].南通大学学报(医学版),2007,27(3):175-177.
[12] 吴爽,魏凤香,李红枝,等.柠檬百里香叶挥发油成分分析及对肝癌细胞毒性作用 [J].中药材,2013,36(5):756-759.
[13] 吴贻琛,万志红,辛绍杰.干细胞移植治疗肝功能衰竭的研究进展及应用前景 [J].肝脏,2015,20(3):251-254.
[14] Mitchell G,Bartlett DW,Fraser TE,et al.Mesotrione:a new selective herbicide for use in maize [J].Pest Manag Sci,2001,57(2):120-128.
[15] Hickey RD,Lillegard JB,Fisher JE,et al.Efficient production of Fah-Null heterozygote pigs by chimeric adeno-associated virus-mediated gene knockout and somatic cell nuclear transfer [J].Hepatology,2011,54(4):1351-1359.
[16] Li L,Zhang Q,Yang H,et al.Fumarylacetoacetate hydrolase knock-out rabbit model for hereditary tyrosinemia typeⅠ[J].J Biol Chem,2017,292(11):4755-4763.
[17] Elgilani F,Mao SA,Glorioso JM,et al.Chronic phenotype characterization of a large animal model of hereditary tyrosinemia typeⅠ[J].Am J Pathol,2017,187(1):33-41.
[18] 黄林华,李西睿,龙川,等.CRISPR/Cas9介导CMAH基因敲除猪创制及繁育分析[J].农业生物技术学报,2018,26(6):1064-1073.
[19] 唐雨婷,高景波,龙川,等.CRISPR/Cas9介导的β4GalNT2基因敲除猪制备 [J].农业生物技术学报,2017,25(10):1697-1705.
[20] Isalan M,Klug A,Choo Y.Comprehensive DNA recognition through concerted interactions from adjacent zinc fingers [J].Biochemistry,1998,37(35):12026-12033.
[21] Pan DK,Zhang L,Zhou YR,et al.Somatic cell nuclear transfer production transfected omega-3 fatty acids to the saturated enzyme gene sFat-1 cloned pig [J].Science in China (Series C:Life Sciences),2009,3:295-302.
[22] 冯书堂.中国实验用小型猪[M].北京:中国农业出版社,2011.
[23] Feng C,Li XR,Cui HT,et al.Highly efficient generation of GGTA1 knockout pigs using a combination of TALEN mRNA and magnetic beads with somatic cell nuclear transfer [J].J Integr Agric,2016,15(7):1540-1549.
[24] Niemann H.Epigenetic reprogramming in mammalian species after SCNT-based cloning[J].Theriogenology,2016,86(1):80-90.
[25] Lai L,Kolber-Simonds D,Park KW,et al.Production of α-1,3 -galactosyltransferase knockout pigs by nuclear transfer cloning [J].Science,2002,295(5557):1089-1092.
[26] Dai Y,Vaught T D,Boone J,et al.Targeted disruption of the alpha1,3-galactosyltransferase gene in cloned pigs [J].Nat Biotechnol,2002,20(3):251-256.
[27] Wu J,Plateroluengo A,Sakurai M,et al.Interspecies chimerism with mammalian pluripotent stem cells [J].Cell,2017,168(3):473-486.
[28] 郑道山,冯冲,朱彦宾,等.利用启动子缺陷型打靶载体敲除五指山小型猪 GGTA1 基因[J].生物技术通讯,2011,22(4):458-462.
[29] Xin J,Yang H,Fan N,et al.Highly efficient generation of GGTA1 biallelic knockout inbred mini-pigs with TALENs [J].PLoS One,2013,8(12):e84250.
[30] Liu HB,Lv PR,He RG,et al.Cloned Guangxi Bama minipig (Sus scrofa) and its offspring have normal reproductive performance [J].Cell Reprogram,2010,12(5):543-550.
[31] 刘芳遐.人肝源性干细胞在Fah-/-Rag2-/-小鼠中生物学行为的研究[D].第二军医大学,2010.
[32] Paulk NK,Wursthorn K,Wang Z,et al.Adeno-associated virus gene repair corrects a mouse model of hereditary tyrosinemia in vivo [J].Hepatology,2010,51(4):1200-1208.
[33] Sinclair KD,Corr SA,Gutierrez CG,et al.Healthy ageing of cloned sheep [J].Nat Commun,2016,7:12359.
[2] Jorquera R,Tanguay RM.The mutagenicity of the tyrosine metabolite,fumarylacetoacetate,is enhanced by glutathione depletion [J].Biochem Biophys Res Commun,1997,232(1):42-48.
[3] Orejuela D,Jorquera R,Bergeron A,et al.Hepatic stress in hereditary tyrosinemia type Ⅰ (HTⅠ) activates the AKT survival pathway in the fah-/- knockout mice model [J].J Hepatol,2008,48(2):308-317.
[4] Qi Z,Wang X,Wei H,et al.Infiltrating neutrophils aggravate metabolic liver failure in fah deficient mice [J].Liver Int,2015,35(3):774-785.
[5] Lindblad B,Lindstedt S,Steen G.On the enzymic defect in hereditary tyrosinemia [J].Proc Natl Acad Sci U S A,1977,74(10):4641-4645.
[6] Hancock ML,Meyer RC,Mistry M,et al.Insulin receptor associates with promoters genome-wide and regulates gene expression [J].Cell,2019,177(3):722-736.e22.
[7] Masurel-Paulet A,Poggi-Bach J,Rolland MO,et al.NTBC treatment in tyrosinaemia type Ⅰ:long-term outcome in French patients [J].J Inherit Metab Dis,2008,31(1):81-87.
[8] Ellis MK,Whitfield AC,Gowans LA,et al.Inhibition of 4-hydroxyphenylpyruvate dioxygenase by 2-(2-nitro-4-trifluoromethylbenzoyl)-cyclohexane-1,3-dione and 2-(2-chloro-4-methanesulfonylbenzoyl)-cyclohexane-1,3-dione[J].Toxicol Appl Pharmacol,1995,133(1):12-19
[9] Holme E,Lindstedt S.Tyrosinaemia type Ⅰ and NTBC (2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione) [J].J Inherit Metab Dis,1998,21(5):507-517.
[10] Molchanov S,Gryffkeller A.Inhibition of 4-hydroxyphenylpyruvate dioxygenase by 2-[2-nitro-4-(trifluoromethyl) benzoyl]-1,3-cyclohexanedione[J].Acta Biochim Pol,2009,56(3):447-454.
[11] 陈钟,陈瑞新,丁义涛,等.新型生物人工肝系统治疗急性肝衰竭犬后猪内源性逆转录病毒传播的实验研究[J].南通大学学报(医学版),2007,27(3):175-177.
[12] 吴爽,魏凤香,李红枝,等.柠檬百里香叶挥发油成分分析及对肝癌细胞毒性作用 [J].中药材,2013,36(5):756-759.
[13] 吴贻琛,万志红,辛绍杰.干细胞移植治疗肝功能衰竭的研究进展及应用前景 [J].肝脏,2015,20(3):251-254.
[14] Mitchell G,Bartlett DW,Fraser TE,et al.Mesotrione:a new selective herbicide for use in maize [J].Pest Manag Sci,2001,57(2):120-128.
[15] Hickey RD,Lillegard JB,Fisher JE,et al.Efficient production of Fah-Null heterozygote pigs by chimeric adeno-associated virus-mediated gene knockout and somatic cell nuclear transfer [J].Hepatology,2011,54(4):1351-1359.
[16] Li L,Zhang Q,Yang H,et al.Fumarylacetoacetate hydrolase knock-out rabbit model for hereditary tyrosinemia typeⅠ[J].J Biol Chem,2017,292(11):4755-4763.
[17] Elgilani F,Mao SA,Glorioso JM,et al.Chronic phenotype characterization of a large animal model of hereditary tyrosinemia typeⅠ[J].Am J Pathol,2017,187(1):33-41.
[18] 黄林华,李西睿,龙川,等.CRISPR/Cas9介导CMAH基因敲除猪创制及繁育分析[J].农业生物技术学报,2018,26(6):1064-1073.
[19] 唐雨婷,高景波,龙川,等.CRISPR/Cas9介导的β4GalNT2基因敲除猪制备 [J].农业生物技术学报,2017,25(10):1697-1705.
[20] Isalan M,Klug A,Choo Y.Comprehensive DNA recognition through concerted interactions from adjacent zinc fingers [J].Biochemistry,1998,37(35):12026-12033.
[21] Pan DK,Zhang L,Zhou YR,et al.Somatic cell nuclear transfer production transfected omega-3 fatty acids to the saturated enzyme gene sFat-1 cloned pig [J].Science in China (Series C:Life Sciences),2009,3:295-302.
[22] 冯书堂.中国实验用小型猪[M].北京:中国农业出版社,2011.
[23] Feng C,Li XR,Cui HT,et al.Highly efficient generation of GGTA1 knockout pigs using a combination of TALEN mRNA and magnetic beads with somatic cell nuclear transfer [J].J Integr Agric,2016,15(7):1540-1549.
[24] Niemann H.Epigenetic reprogramming in mammalian species after SCNT-based cloning[J].Theriogenology,2016,86(1):80-90.
[25] Lai L,Kolber-Simonds D,Park KW,et al.Production of α-1,3 -galactosyltransferase knockout pigs by nuclear transfer cloning [J].Science,2002,295(5557):1089-1092.
[26] Dai Y,Vaught T D,Boone J,et al.Targeted disruption of the alpha1,3-galactosyltransferase gene in cloned pigs [J].Nat Biotechnol,2002,20(3):251-256.
[27] Wu J,Plateroluengo A,Sakurai M,et al.Interspecies chimerism with mammalian pluripotent stem cells [J].Cell,2017,168(3):473-486.
[28] 郑道山,冯冲,朱彦宾,等.利用启动子缺陷型打靶载体敲除五指山小型猪 GGTA1 基因[J].生物技术通讯,2011,22(4):458-462.
[29] Xin J,Yang H,Fan N,et al.Highly efficient generation of GGTA1 biallelic knockout inbred mini-pigs with TALENs [J].PLoS One,2013,8(12):e84250.
[30] Liu HB,Lv PR,He RG,et al.Cloned Guangxi Bama minipig (Sus scrofa) and its offspring have normal reproductive performance [J].Cell Reprogram,2010,12(5):543-550.
[31] 刘芳遐.人肝源性干细胞在Fah-/-Rag2-/-小鼠中生物学行为的研究[D].第二军医大学,2010.
[32] Paulk NK,Wursthorn K,Wang Z,et al.Adeno-associated virus gene repair corrects a mouse model of hereditary tyrosinemia in vivo [J].Hepatology,2010,51(4):1200-1208.
[33] Sinclair KD,Corr SA,Gutierrez CG,et al.Healthy ageing of cloned sheep [J].Nat Commun,2016,7:12359.