GTKO/hCD55基因编辑异种器官移植供体猪的构建及功能验证

doi:10.12300/j.issn.1674-5817.2025.024

摘要/Abstract

摘要：

目的开发GTKO/hCD55基因编辑异种器官移植供体猪并进行功能验证。方法利用CRISPR/Cas9、piggyBac（PB）转座子技术和体细胞克隆技术，构建GTKO/hCD55基因编辑的滇南小耳猪。再通过PCR、Sanger测序、细胞流式、免疫荧光、重亚硫酸盐测序分析GTKO/hCD55猪的表型及功能。结果将载体转染至成纤维细胞后，经药物筛选获得48个单细胞克隆点，挑选两个单细胞克隆点进行体细胞克隆，获得两个33 d的胎猪。F01胎猪的GGTA1基因型为-17 bp和WT，F02胎猪的GGTA1基因型为-26 bp/+2 bp和-3 bp。两个胎猪的hCD55 mRNA表达量均与野生型（WT）猪有显著差异，选择F02胎猪作为供体细胞来源进行重克隆后获得11头成活仔猪，鉴定均为GTKO/hCD55基因编辑猪。这些猪αGal抗原表达缺失，但hCD55出现弱表达或不表达的情况。因此，对hCD55基因的CpG岛进行甲基化分析，结果表明在不表达hCD55的肾脏组织中hCD55基因的CpG岛被完全甲基化，而表达hCD55的肾脏组织中hCD55基因的CpG岛未被甲基化或部分被甲基化。对hCD55基因的CpG岛进行密码子优化降低CG含量后，可实现hCD55基因的稳定表达。此外，抗原抗体结合实验表明人IgM结合到GTKO/hCD55基因编辑猪成纤维细胞的数量显著小于WT猪；补体依赖的细胞毒性实验表明GTKO/hCD55猪的成纤维细胞成活率明显高于WT猪。结论成功获得了GTKO/hCD55基因编辑异种器官移植供体猪，通过优化密码子降低过表达基因的CG含量，可有效避免甲基化导致的基因表达沉默，本研究为异种器官移植研究提供了可用的供体猪，并为供体猪的开发提供了借鉴。

关键词: 异种器官移植, 滇南小耳猪, GGTA1, hCD55

Abstract:

Objective To develop GTKO/hCD55 gene-edited xenotransplant donor pigs and identify their phenotype. Methods In this study, CRISPR/Cas9, piggyBac (PB) transposon technology and somatic cell cloning technology were used to construct GTKO/hCD55gene-edited Dinnan miniature pigs. The phenotype and function of GTKO/hCD55 pigs were analyzed by PCR, Sanger sequencing, cell flow cytometry, immunofluorescence and bisulfite sequencing. Base on pig fibroblasts with O blood type, the GTKO/hCD55 gene edited Diannan miniature pigs were constructed by using CRISPR/Cas9, piggyBac (PB) transposon technology and somatic cell cloning technology. Then, the phenotype and function of GTKO/hCD55 pigs were analyzed by PCR, Sanger sequencing, cell flow cytometry, immunofluorescence, and bisulfite sequencing. Results After transfection of vectors into fibroblasts, 48 single-cell colonies were obtained through drug screening. Two single-cell colonies were selected for cloning, resulting in two 33 day-old fetal pigs. The GGTA1 genotypes of fetal pig F01 are -17 bp and WT, while the GGTA1 genotypes of fetal pig F02 are -26 bp/+2 bp and -3 bp. The hCD55 mRNA expression levels of both fetal pigs were significantly higher than those of wild-type (WT) pigs. The fetal pig F02 was selected as the donor cell source for recloning, 11 surviving piglets were obtained. Identifing them as GTKO/hCD55 gene edited pigs. Them was found that the expression of αGal antigen was absent in GTKO/hCD55 gene edited pigs, but hCD55 showed weak or no expression. Therefore, methylation analysis of the CpG island of the hCD55 gene showed that the CpG island of the hCD55 gene was completely methylated in kidney tissue that did not express hCD55, while the CpG island of the hCD55 gene was not methylated or partially methylated in kidney tissue that expressed hCD55. Moreover, codon optimization of the CpG island of the hCD55 gene to reduce CG content can achieve stable expression of the hCD55 gene. In addition, antigen antibody binding experiments showed that the number of human IgM binding to GTKO/hCD55 gene edited pig fibroblasts was significantly lower than that of WT pigs; The complement dependent cytotoxicity experiment showed that the survival rate of fibroblasts in GTKO/hCD55 pigs was significantly higher than that in WT pigs. Conclusion We successfully obtained GTKO/hCD55 gene edited xenotransplant donor pigs. Transgene expression silencing caused by methylation can be optimized by reducing the CG content. This study provides a usable donor pig for xenotransplantation research and provides reference for the development of donor pigs.

Key words: Xenotransplantation, Diannan miniature pigs, GGTA1, hCD55

中图分类号:

Q95-33

王娇祥,张璐,陈姝含,等.GTKO/hCD55基因编辑异种器官移植供体猪的构建及功能验证[J]. 实验动物与比较医学.. DOI: 10.12300/j.issn.1674-5817.2025.024.

WANG Jiaoxiang,ZHANG Lu,CHEN Shuhan,et al. Construction and Functional Validation of GTKO/hCD55 Gene-Edited Xenotransplant Donor Pigs[J]. Laboratory Animal and Comparative Medicine. DOI: 10.12300/j.issn.1674-5817.2025.024.

图/表 7

图1 GTKO/hCD55基因编辑阳性单细胞克隆点的筛选注：A为GGTA1基因敲除和hCD55基因过表达载体的示意图；B为阳性单细胞克隆点的GGTA1靶区域基因型；C为48个单细胞克隆点hCD55基因的PCR鉴定。

Figure 1 Screening of single-cell clone sites positive for GTKO/hCD55 gene editingNote：A， Schematic representation of the GGTA1 gene knockout and hCD55 gene overexpression vectors. B， GGTA1 target region genotype of positive single-cell clone sites； C， PCR identification of the hCD55 gene from 48 single-cell clone sites.

表1 胚胎移植和克隆仔猪的产生

Table 1 The embryo transfer and generation of cloned piglets

No.	ID	Body weight (g)	Status at birth	Survival time (day)	Recipients
1	DN-GTKO-hCD55F02P01	500	healthy	3	P516
2	DN-GTKO-hCD55F02P02	400	healthy	1	P516
3	DN-GTKO-hCD55F02P03	720	stillbirth		P452
4	DN-GTKO-hCD55F02P04	380	stillbirth
5	DN-GTKO-hCD55F02P05	620	palate	4
6	DN-GTKO-hCD55F02P06	200	mummy		P511
7	DN-GTKO-hCD55F02P07	560	stillbirth
8	DN-GTKO-hCD55F02P08	300	mummy
9	DN-GTKO-hCD55F02P09	400	mummy
10	DN-GTKO-hCD55F02P10	480	stillbirth		P443
11	DN-GTKO-hCD55F02P11	560	healthy	2
12	DN-GTKO-hCD55F02P12	580	healthy	3
13	DN-GTKO-hCD55F02P13	520	healthy	1
14	DN-GTKO-hCD55F02P14	260	weaker	2
15	DN-GTKO-hCD55F02P15	320	stillbirth
16	DN-GTKO-hCD55F02P16	620	healthy	2
17	DN-GTKO-hCD55F02P17	740	healthy	1
18	DN-GTKO-hCD55F02P18	560	stillbirth		P525
19	DN-GTKO-hCD55F02P19	540	healthy	21
20	DN-GTKO-hCD55F02P20	660	healthy	206

图2 GTKO/hCD55基因编辑克隆胎猪的鉴定注：A为2个33天的克隆胎猪；B为猪性别鉴定；C为胎猪血型鉴定；D为胎猪hCD55的PCR鉴定结果；E为胎猪GGTA1基因靶区域的PCR结果；F为胎猪GGTA1基因靶区域的基因型；G为胎猪组织hCD55的mRNA表达水平；H为胎猪成纤维细胞中αGal和hCD55表达的流式结果。

Figure 2 Identification of fetal pigs with GTKO/hCD55 gene-edited clonesNote：A， two cloned fetal pigs at 33 days； B， pig sex identification； C， Fetal pig blood typing； D， PCR identification of fetal pig hCD55； E， PCR results of the target region of the fetal pig GGTA1 gene； F， genotype of the GGTA1 target region in fetal pigs； G， hCD55 mRNA expression level in fetal pig tissues； H， Flow results of αGal and hCD55 expression in fetal porcine fibroblasts.

图3 猪GTKO/hCD55基因编辑仔猪的鉴定注：A为GTKO/hCD55克隆仔猪照片；B为仔猪hCD55的PCR鉴定结果；C为仔猪GGTA1基因靶区域的PCR结果；D为仔猪GGTA1基因靶区域的基因型；E为仔猪成纤维细胞中αGal和hCD55表达的流式结果；F为仔猪肾脏免疫荧光结果。

Figure 3 Identification of porcine GTKO/hCD55 gene-edited pigletsNote：A， Photo of cloned piglets of GTKO/hCD55； B， PCR identification of hCD55 in piglets； C， PCR results of the target region of GGTA1 gene in piglets； D， genotype of GGTA1 target region in piglets； E， Flow cytometry results for αGal and hCD55 expression in piglet-derived fibroblasts； F， immunofluorescence results of piglet kidneys.

图4 细胞克隆点、胎猪、克隆个体中hCD55基因甲基化水平注：A为hCD55基因上的CpG岛示意图；B为混合克隆点C35&C45、克隆胎猪F02成纤维细胞、克隆仔猪（P07、P13、P12、P14、P17）肾脏组织中的hCD55的甲基化水平。

Figure 4 Methylation levels of the hCD55 gene in cell clone sites， fetal pigs， and cloned individualsNote：A， Schematic representation of CpG islands on the hCD55 gene； B， Methylation levels of hCD55 in mixed clone sites C35&C45， cloned fetal pig F02 fibroblasts， and cloned piglet （P07， P13， P12， P14， P17） kidney tissues.

图5 密码子优化前后hCD55在成纤维细胞中的表达水平注：A为密码子优化前后hCD55基因CpG岛区域的Sanger测序结果；B为优化前后的载体转染猪成纤维细胞，流式检测hCD55在细胞中的表达水平（3次重复实验）。

Figure 5 Expression Levels of hCD55 in Fibroblasts before and after codon optimizationNote：A， Sanger sequencing results of CpG island region of hCD55 gene before and after codon optimization； B， Porcine fibroblasts were transfected with the optimized and optimized vectors， and the expression level of hCD55 in the cells was detected by flow cytometry （triplicate experiments）.

图6 GTKO/hCD55基因编辑猪的抗免疫排斥效果评价注：A为混合人血清孵育猪成纤维细胞后，人IgG和IgM抗体与猪成纤维细胞的结合程度；B为混合人血清孵育猪成纤维细胞后，人补体对猪细胞的杀伤程度。

Figure 6 Evaluation of anti-immune rejection efficacy in GTKO/hCD55 gene-edited pigsNote：A， extent of binding of human IgG and IgM antibodies to porcine fibroblasts after incubation with mixed human serum； B， extent of killing of porcine cells by human complement after incubation of porcine fibroblasts with mixed human serum.

参考文献 27

1	ALI A, KEMTER E, WOLF E. Advances in organ and tissue xenotransplantation[J]. Annual Review of Animal Biosciences, 2024, 12: 369–390. DOI:10.1146/annurev-animal-021122-102606 .
2	SINGH A K, GOERLICH C E, ZHANG T, et al. Genetically engineered pig heart transplantation in non-human primates[J]. Communications Medicine, 2025, 5: 6. DOI:10.1038/s43856-025-00731-y .
3	EISENSON D, HISADOME Y, SANTILLAN M, et al. Consistent survival in consecutive cases of life-supporting porcine kidney xenotransplantation using 10ge source pigs[J]. Nature Communications, 2024, 15: 3361. DOI:10.1038/s41467-024-47679-6 .
4	ANAND R P, LAYER J V, HEJA D, et al. Design and testing of a humanized porcine donor for xenotransplantation[J]. Nature, 2023, 622(7982): 393–401. DOI:10.1038/s41586-023-06594-4 .
5	MOHIUDDIN M M, SINGH A K, SCOBIE L, et al. Graft dysfunction in compassionate use of genetically engineered pig-to-human cardiac xenotransplantation: a case report[J]. Lancet, 2023, 402(10399): 397–410. DOI:10.1016/S0140-6736(23)00775-4 .
6	GRIFFITH B P, GOERLICH C E, SINGH A K, et al. Genetically modified porcine-to-human cardiac xenotransplantation[J]. The New England journal of Medicine, 2022, 387(1): 35–44. DOI:10.1056/NEJMoa2201422 .
7	PAN W, ZHANG W, ZHENG B, et al. Cellular dynamics in pig-to-human kidney xenotransplantation[J]. Med, 2024, 5(8): 1016-1029.e4. DOI:10.1016/j.medj.2024.05.003 .
8	KAWAI T, WILLIAMS W W, ELIAS N, et al. Xenotransplantation of a porcine kidney for end-stage kidney disease[J]. The New England Journal of Medicine, 2025. DOI:10.1056/NEJMoa2412747 .
9	MALLAPATY S. First pig-to-human liver transplant recipient "doing very well"[J]. Nature, 2024, 630(8015): 18. DOI:10.1038/d41586-024-01613-4 .
10	KUWAKI K, Y-L TSENG, DOR F J M F, et al. Heart transplantation in baboons using alpha1,3-galactosyltransferase gene-knockout pigs as donors: initial experience[J]. Nature Medicine, 2005, 11(1): 29–31. DOI:10.1038/nm1171 .
11	AZIMZADEH A M, KELISHADI S S, EZZELARAB M B, et al. Early graft failure of galtko pig organs in baboons is reduced by expression of a human complement pathway-regulatory protein[J]. Xenotransplantation, 2015, 22(4): 310–316. DOI:10.1111/xen.12176 .
12	MOHIUDDIN M M, GOERLICH C E, SINGH A K, et al. Progressive genetic modifications of porcine cardiac xenografts extend survival to 9 months[J]. Xenotransplantation, 2022, 29(3): e12744. DOI:10.1111/xen.12744 .
13	CHABAN R, MCGRATH G, HABIBABADY Z, et al. Increased human complement pathway regulatory protein gene dose is associated with increased endothelial expression and prolonged survival during ex-vivo perfusion of gtko pig lungs with human blood[J]. Xenotransplantation, 2023, 30(4): e12812. DOI:10.1111/xen.12812 .
14	WARD T, PIPKIN P A, CLARKSON N A, et al. Decay-accelerating factor cd55 is identified as the receptor for echovirus 7 using celics, a rapid immuno-focal cloning method[J]. The EMBO Journal, 1994, 13(21): 5070–5074. DOI:10.1002/j.1460-2075.1994.tb06836.x .
15	LUKACIK P, ROVERSI P, WHITE J, et al. Complement regulation at the molecular level: the structure of decay-accelerating factor[J]. Proceedings of the National Academy of Sciences of the United States of America, 2004, 101(5): 1279–1284. DOI:10.1073/pnas.0307200101 .
16	MURAKAMI H, NAGASHIMA H, TAKAHAGI Y, et al. Transgenic pigs expressing human decay-accelerating factor regulated by porcine mcp gene promoter[J]. Molecular Reproduction and Development, 2002, 61(3): 302–311. DOI:10.1002/mrd.10043 .
17	KIM S C, MATHEWS D V, BREEDEN C P, et al. Long-term survival of pig-to-rhesus macaque renal xenografts is dependent on cd4 t cell depletion[J]. American Journal of Transplantation: Official Journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 2019, 19(8): 2174–2185. DOI:10.1111/ajt.15329 .
18	李欣, 潘登科, 周佳, 等. 表达人源补体调节蛋白hCD55对异种胰岛移植的保护作用[J]. 器官移植, 2022, 13(4): 475–482. DOI:10.3969/j.issn.1674-7445.2022.04.010 .
19	夏强兵, 冯豪, 王璐, 等. GTKO/hcd55基因工程猪到人异种移植cdc技术的探讨[J]. 中华器官移植杂志, 2022, 43(06): 364–369. DOI:10.3760/cma.j.cn421203-20220505-00096 .
20	YUE Y, XU W, KAN Y, et al. Extensive germline genome engineering in pigs[J]. Nature Biomedical Engineering, 2021, 5(2): 134–143. DOI:10.1038/s41551-020-00613-9 .
21	LETI F, LLACI L, MALENICA I, et al. Chapter 13: methods for cpg methylation array profiling via bisulfite conversion[J]. Methods in molecular biology (Clifton, N.J.), 2018, 1706: 233–254. DOI:10.1007/978-1-4939-7471-9_13 .
22	MOAZAMI N, STERN J M, KHALIL K, et al. Pig-to-human heart xenotransplantation in two recently deceased human recipients[J]. Nature Medicine, 2023, 29(8): 1989–1997. DOI:10.1038/s41591-023-02471-9 .
23	LOCKE J E, KUMAR V, ANDERSON D, et al. Normal graft function after pig-to-human kidney xenotransplant[J]. JAMA Surgery, 2023, 158(10): 1106–1108. DOI:10.1001/jamasurg.2023.2774 .
24	WANG J, XU K, LIU T, et al. Production and functional verification of 8-gene (ggta1, cmah, β4galnt2, hcd46, hcd55, hcd59, htbm, hcd39)-edited donor pigs for xenotransplantation[J]. Cell Proliferation, 2025: e70028. DOI:10.1111/cpr.70028 .
25	MANOOK M, OLASO D, ANWAR I, et al. Prolonged xenokidney graft survival in sensitized nhp recipients by expression of multiple human transgenes in a triple knockout pig[J]. Science translational Medicine, 2024, 16(751): eadk6152. DOI:10.1126/scitranslmed.adk6152 .
26	YANG C, WEI Y, LI X, et al. Production of four-gene (gtko/hcd55/htbm/hcd39)-edited donor pigs and kidney xenotransplantation[J]. Xenotransplantation, 2024, 31(4): e12881. DOI:10.1111/xen.12881 .
27	ZHAO H, LI Y, WIRIYAHDAMRONG T, et al. Improved production of gtko/hcd55/hcd59 triple-gene-modified diannan miniature pigs for xenotransplantation by recloning[J]. Transgenic Research, 2020, 29(3): 369–379. DOI:10.1007/s11248-020-00201-2 .

[1]	朱婵, 张栋梁, 赵德莉, 石雪琴, 千磊, 张玄, 金艳, 段伟, 戚若晨, 刘超华, 杨薛康, 韩军涛, 潘登科. 基因编辑猪-猴异种组织器官移植围手术期动物护理[J]. 实验动物与比较医学, 2024, 44(5): 495-501.
[2]	王娇祥, 王艳, 胡珂, 徐凯祥, 魏太云, 角德灵, 赵恒, 赵红业, 魏红江. 猪血型PCR鉴定方法的建立[J]. 实验动物与比较医学, 2023, 43(6): 585-594.
[3]	刘荣辉, 卢丽仪, 陶璇, 于辉, 李华. 滇南小耳猪与云南野猪SLA-DQA基因的多态性比较[J]. 实验动物与比较医学, 2011, 31(5): 360-364.
[4]	倪丽菊, 谢建云, 胡建华. 云南滇南小耳猪与广西巴马小型猪mtDNA D-Loop的初步研究[J]. 实验动物与比较医学, 2011, 31(3): 207-210.