在已获得α1,3-半乳糖基转移酶(al,3-galactosyltransferase,GGTAl)基因双敲除猪的基础上,建立GGTAl双敲除猪和普通猪的耳成纤维细胞系。利用正常人血清(Normal Human Serum,NHS)、灭活人血清(Heat-Inactivated Human Serum,HIA-HS)和...在已获得α1,3-半乳糖基转移酶(al,3-galactosyltransferase,GGTAl)基因双敲除猪的基础上,建立GGTAl双敲除猪和普通猪的耳成纤维细胞系。利用正常人血清(Normal Human Serum,NHS)、灭活人血清(Heat-Inactivated Human Serum,HIA-HS)和胎牛血清(Fetal Bovine Serum,FBS)处理经钙黄绿素(CalceinAM)染色细胞。通过流式细胞仪检测细胞荧光强度和凋亡情况。结果显示,经NHS处理的GGTAl双敲除猪细胞死亡率(<10%)比普通猪细胞死亡率(>40%)显著降低,证明细胞表面的α1,3-半乳糖基(α1,3-galactosyl,a1,3Gal)明显减少,在异种器官移植研究中抑制免疫排斥反应起到一定的作用。展开更多
Clinical xenotransplantations have been hampered by human preformed antibody-mediated damage of the xenografts.To overcome biological incompatibility between pigs and humans,one strategy is to remove the major antigen...Clinical xenotransplantations have been hampered by human preformed antibody-mediated damage of the xenografts.To overcome biological incompatibility between pigs and humans,one strategy is to remove the major antigens[Gal,Neu5 Gc,and Sd(a)]present on pig cells and tissues.Triple gene(GGTAI,CMAH,and β4 GalNT2)knockout(TKO)pigs were produced in our laboratory by CRISPR-Cas9 targeting.To investigate the antigenicity reduction in the TKO pigs,the expression levels of these three xenoantigens in the cornea,heart,liver,spleen,lung,kidney,and pancreas tissues were examined.The level of human IgG/IgM binding to those tissues was also investigated,with wildtype pig tissues as control.The results showed that aGal,Neu5 Gc,and Sd(a)were markedly positive in all the examined tissues in wildtype pigs but barely detected in TKO pigs.Compared to wildtype pigs,the liver,spleen,and pancreas of TKO pigs showed comparable levels of human IgG and IgM binding,whereas corneas,heart,lung,and kidney of TKO pigs exhibited significantly reduced human IgG and IgM binding.These results indicate that the antigenicity of TKO pig is significantly reduced and the remaining xenoantigens on porcine tissues can be eliminated via a gene targeting approach.展开更多
制备α1,3-半乳糖基转移酶(αl,3-galactosyltransferase,GGTA1)基因缺失的五指山小型猪,为我国异种器官移植研究奠定基础。在已经获得的GGTA1单等位基因敲除(GGTA+/-)五指山小型猪基础上,建立GGTA1+/-猪耳成纤维细胞系。利用特异性结合...制备α1,3-半乳糖基转移酶(αl,3-galactosyltransferase,GGTA1)基因缺失的五指山小型猪,为我国异种器官移植研究奠定基础。在已经获得的GGTA1单等位基因敲除(GGTA+/-)五指山小型猪基础上,建立GGTA1+/-猪耳成纤维细胞系。利用特异性结合α1,3Gal的药物GSI-B4联合免疫磁珠筛选,成功分离出自发杂合性缺失(Loss of heterozygosity,LOH)的双等位基因敲除(GGTA1-/-)耳成纤维细胞。单细胞克隆培养与PCR鉴定后,以GGTA1-/-细胞为核供体,体外成熟的猪卵母细胞为核受体构建克隆胚胎并移植。先后将3 122枚重构胚移植到13头受体母猪,其中4头怀孕,产仔12头。采用PCR和Southern blotting进行GGTA1基因型检测,11头为GGTA1-/-猪;流式细胞术分析表明,GGTA1-/-仔猪耳成纤维细胞不表达α1,3Gal。获得能克服异种器官超急性排斥反应(HAR)的GGTA1-/-猪,不仅为异种器官供体的进一步基因修饰提供了平台,也为异种移植临床前研究提供了宝贵资源。展开更多
Genetically modified pigs are valuable models of human disease and donors of xenotransplanted organs.Conventional gene targeting in pig somatic cells is extremely inefficient.Zinc-finger nuclease(ZFN)technology has be...Genetically modified pigs are valuable models of human disease and donors of xenotransplanted organs.Conventional gene targeting in pig somatic cells is extremely inefficient.Zinc-finger nuclease(ZFN)technology has been shown to be a powerful tool for efficiently inducing mutations in the genome.However,ZFN-mediated targeting in pigs has rarely been achieved.Here,we used ZFNs to knock out the porcineα-1,3-galactosyl-transferase(GGTA1)gene,which generates Gal epitopes that trigger hyperacute immune rejection in pig-to-human transplantation.Primary pig fibroblasts were transfected with ZFNs targeting the coding region of GGTA1.Eighteen mono-allelic and four biallelic knockout cell clones were obtained after drug selection with efficiencies of 23.4%and 5.2%,respectively.The biallelic cells were used to produce cloned pigs via somatic cell nuclear transfer(SCNT).Three GGTA1 null piglets were born,and one knockout primary fibroblast cell line was established from a cloned fetus.Gal epitopes on GGTA1 null pig cells were completely eliminated from the cell membrane.Functionally,GGTA1 knockout cells were protected from complement-mediated immune attacks when incubated with human serum.This study demonstrated that ZFN is an efficient tool in creating gene-modified pigs.GGTA1 null pigs and GGTA1 null fetal fibroblasts would benefit research and pig-to-human transplantation.展开更多
文摘在已获得α1,3-半乳糖基转移酶(al,3-galactosyltransferase,GGTAl)基因双敲除猪的基础上,建立GGTAl双敲除猪和普通猪的耳成纤维细胞系。利用正常人血清(Normal Human Serum,NHS)、灭活人血清(Heat-Inactivated Human Serum,HIA-HS)和胎牛血清(Fetal Bovine Serum,FBS)处理经钙黄绿素(CalceinAM)染色细胞。通过流式细胞仪检测细胞荧光强度和凋亡情况。结果显示,经NHS处理的GGTAl双敲除猪细胞死亡率(<10%)比普通猪细胞死亡率(>40%)显著降低,证明细胞表面的α1,3-半乳糖基(α1,3-galactosyl,a1,3Gal)明显减少,在异种器官移植研究中抑制免疫排斥反应起到一定的作用。
基金National Natural Science Foundation of China(81941001)Natural Science Foundation of Guangdong Province,China(2019A1515110283)+1 种基金Science and Technology Planning Project of Jiangmen,Guangdong Province,China(2021030101220004887)Science Foundation for Young Faculty of Wuyi University(2019TD05)。
基金supported by grants from the National Natural Science Foundation of China(81570402&31701283)the National Key R&D Program of China(2017YFC1103701&2017YFC1103702)+3 种基金the Jiangsu Key Laboratory of Xenotransplantation(BM2012116)the Sanming Project of Medicine in Shenzhenthe Fund for High Level Medical Discipline Construction of Shenzhen(2016031638)the Shenzhen Foundation of Science and Technology(JCYJ20160229204849975&GCZX2015043017281705)
文摘Clinical xenotransplantations have been hampered by human preformed antibody-mediated damage of the xenografts.To overcome biological incompatibility between pigs and humans,one strategy is to remove the major antigens[Gal,Neu5 Gc,and Sd(a)]present on pig cells and tissues.Triple gene(GGTAI,CMAH,and β4 GalNT2)knockout(TKO)pigs were produced in our laboratory by CRISPR-Cas9 targeting.To investigate the antigenicity reduction in the TKO pigs,the expression levels of these three xenoantigens in the cornea,heart,liver,spleen,lung,kidney,and pancreas tissues were examined.The level of human IgG/IgM binding to those tissues was also investigated,with wildtype pig tissues as control.The results showed that aGal,Neu5 Gc,and Sd(a)were markedly positive in all the examined tissues in wildtype pigs but barely detected in TKO pigs.Compared to wildtype pigs,the liver,spleen,and pancreas of TKO pigs showed comparable levels of human IgG and IgM binding,whereas corneas,heart,lung,and kidney of TKO pigs exhibited significantly reduced human IgG and IgM binding.These results indicate that the antigenicity of TKO pig is significantly reduced and the remaining xenoantigens on porcine tissues can be eliminated via a gene targeting approach.
文摘制备α1,3-半乳糖基转移酶(αl,3-galactosyltransferase,GGTA1)基因缺失的五指山小型猪,为我国异种器官移植研究奠定基础。在已经获得的GGTA1单等位基因敲除(GGTA+/-)五指山小型猪基础上,建立GGTA1+/-猪耳成纤维细胞系。利用特异性结合α1,3Gal的药物GSI-B4联合免疫磁珠筛选,成功分离出自发杂合性缺失(Loss of heterozygosity,LOH)的双等位基因敲除(GGTA1-/-)耳成纤维细胞。单细胞克隆培养与PCR鉴定后,以GGTA1-/-细胞为核供体,体外成熟的猪卵母细胞为核受体构建克隆胚胎并移植。先后将3 122枚重构胚移植到13头受体母猪,其中4头怀孕,产仔12头。采用PCR和Southern blotting进行GGTA1基因型检测,11头为GGTA1-/-猪;流式细胞术分析表明,GGTA1-/-仔猪耳成纤维细胞不表达α1,3Gal。获得能克服异种器官超急性排斥反应(HAR)的GGTA1-/-猪,不仅为异种器官供体的进一步基因修饰提供了平台,也为异种移植临床前研究提供了宝贵资源。
基金supported by grants from Ministry of Science and Technology of China(2011CBA01001,2012AA020503)the National Science Fund for Distinguished Young Scholars(31025016)+4 种基金the National Natural Science Foundation of China(31271577)Novel Agricultural Variety Breeding Project of Zhejiang Province(2012C12906-8)the Fundamental Research Funds for the Central Universitiesthe Key Construction Pro-gram of the National"985"Project(118000+193411801/006)the Research Fund for the Doctoral Program of Higher Education of China(20120101110089)
文摘Genetically modified pigs are valuable models of human disease and donors of xenotransplanted organs.Conventional gene targeting in pig somatic cells is extremely inefficient.Zinc-finger nuclease(ZFN)technology has been shown to be a powerful tool for efficiently inducing mutations in the genome.However,ZFN-mediated targeting in pigs has rarely been achieved.Here,we used ZFNs to knock out the porcineα-1,3-galactosyl-transferase(GGTA1)gene,which generates Gal epitopes that trigger hyperacute immune rejection in pig-to-human transplantation.Primary pig fibroblasts were transfected with ZFNs targeting the coding region of GGTA1.Eighteen mono-allelic and four biallelic knockout cell clones were obtained after drug selection with efficiencies of 23.4%and 5.2%,respectively.The biallelic cells were used to produce cloned pigs via somatic cell nuclear transfer(SCNT).Three GGTA1 null piglets were born,and one knockout primary fibroblast cell line was established from a cloned fetus.Gal epitopes on GGTA1 null pig cells were completely eliminated from the cell membrane.Functionally,GGTA1 knockout cells were protected from complement-mediated immune attacks when incubated with human serum.This study demonstrated that ZFN is an efficient tool in creating gene-modified pigs.GGTA1 null pigs and GGTA1 null fetal fibroblasts would benefit research and pig-to-human transplantation.
