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

实验动物与比较医学 ›› 2025, Vol. 45 ›› Issue (4): 379-392.DOI: 10.12300/j.issn.1674-5817.2025.024

• 人类疾病动物模型 • 下一篇

GTKO/hCD55基因编辑异种器官移植供体猪的构建及功能验证

王娇祥¹^,²^,³, 张璐¹^,²^,⁴, 陈姝含¹^,²^,⁴, 角德灵¹^,²^,⁴, 赵恒¹^,²^,⁴, 魏太云¹^,²^,⁴, 郭建雄¹^,²^,⁴, 徐凯祥¹^,²^,⁴, 魏红江¹^,²^,³^,⁴()()

^1.云南省小型猪基因编辑与异种器官移植重点实验室, 昆明 650201
^2.云南省异种器官移植工程研究中心, 昆明 650201
^3.云南农业大学动物科学技术学院, 昆明 650201
^4.云南农业大学动物医学院, 昆明 650201

收稿日期:2025-02-20 修回日期:2025-04-27 出版日期:2025-08-25 发布日期:2025-09-01
通讯作者: 魏红江（1971—），男，博士，教授，主要从事动物基因编辑与体细胞克隆技术研究。E-mail： hongjiangwei@126.com。ORCID： 0000-0002-5663-1093
作者简介:王娇祥（1996—），女，博士研究生，主要从事基因编辑研究。E-mail：jiaoxiangwang2021@126.com；
张璐（2000—），女，硕士研究生，主要从事基础兽医学研究。E-mail：3077289149@qq.com；
陈姝含（1996—），女，硕士研究生，主要从事基础兽医学研究。E-mail：chenshuhan090203@163.com
基金资助:
国家重点研发计划-干细胞及转化研究重点专项“异种移植用人源化基因编辑供体猪的构建及临床前研究”(2019YFA0110700)

Construction and Functional Validation of GTKO/hCD55 Gene-Edited Xenotransplant Donor Pigs

WANG Jiaoxiang¹^,²^,³, ZHANG Lu¹^,²^,⁴, CHEN Shuhan¹^,²^,⁴, JIAO Deling¹^,²^,⁴, ZHAO Heng¹^,²^,⁴, WEI Taiyun¹^,²^,⁴, GUO Jianxiong¹^,²^,⁴, XU Kaixiang¹^,²^,⁴, WEI Hongjiang¹^,²^,³^,⁴()()

^1.Yunnan Province Key Laboratory for Porcine Gene Editing and Xenotransplantation, Kunming 650201, China
^2.Yunnan Province Xenotransplantation Research Engineering Center, Kunming 650201, China
^3.Faculty of Animal Science and Technology, Yunnan Agricultural University, Kunming 650201, China
^4.College of Veterinary Medicine, Yunnan Agricultural University, Kunming 650201, China

Received:2025-02-20 Revised:2025-04-27 Published:2025-08-25 Online:2025-09-01
Contact: WEI Hongjiang (ORCID: 0000-0002-5663-1093), E-mail: hongjiangwei@126.com

摘要/Abstract

摘要：

目的开发α-1，3半乳糖苷转移酶基因敲除（α-1，3-galactosyltransferase gene-knockout，GTKO）/人CD55（human CD55，hCD55）转基因的基因编辑异种器官移植供体猪并进行功能验证。方法利用成簇的规律间隔的短回文重复序列（clustered regularly interspaced short palindromic repeats，CRISPR）/CRISPR相关蛋白核酸酶9（CRISPR-associated nuclease 9，Cas9）、PiggyBac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，通过Sanger测序、实时荧光定量PCR、流式细胞术、免疫荧光实验、重亚硫酸盐测序、抗原抗体结合实验和补体依赖的细胞毒性实验分析其表型及功能。结果将基因编辑载体PX458和PiggyBac转染至野生型胎猪成纤维细胞后，经嘌呤霉素药物筛选获得48个单细胞克隆，挑选2个单细胞克隆进行体细胞克隆，获得2个妊娠33 d的胎猪。F01胎猪的α-1，3-半乳糖基转移酶（α-1，3-galactosyltransferase，GGTA1）基因型为-17 bp和野生型（wild type，WT），F02胎猪的GGTA1基因型为-26 bp/+2 bp和-3 bp。两个胎猪的hCD55 mRNA表达量均显著高于WT猪（P＜0.01），以F02胎猪作为供体细胞来源进行连续克隆后获得11头存活仔猪，鉴定均为GTKO/hCD55基因编辑猪。这些猪的α-Gal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岛被高甲基化，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（P＜0.01）；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（P＜0.01）。结论成功获得了GTKO/hCD55基因编辑异种器官移植供体猪，并通过密码子优化降低了hCD55基因CpG岛的CG含量，从而有效避免甲基化导致的基因表达沉默。本研究可为供体猪的开发提供借鉴，将指导后续异种器官移植研究。

关键词: 异种器官移植, 滇南小耳猪, α-1, 3-半乳糖基转移酶, 人CD55

Abstract:

Objective To develop GTKO (α-1,3-galactosyltransferase gene-knockout, GTKO)/hCD55 (human CD55) gene-edited xenotransplant donor pigs and verify their function. Methods In this study, CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated nuclease 9), PiggyBac transposon technology and somatic cell nuclear transfer technology were used to construct GTKO/hCD55 gene-edited Diannan miniature pigs. The phenotype and function of GTKO/hCD55 pigs were analyzed by Sanger sequencing, real-time fluorescence quantitative PCR, flow cytometry, immunofluorescence, bisulfite sequencing, antigen-antibody binding assays, and complement-dependent cytotoxicity assays. Results After transfection of PX458 and PiggyBac gene editing vectors into wild-type fetal pig fibroblasts, 48 single-cell colonies were obtained through puromycin drug screening. Two single-cell colonies were selected for somatic cell nuclear transfer, resulting in two fetal pigs at 33 days of gestation. The GGTA1(α-1,3-galactosyltransferase) genotypes of fetal pig F01 were -17 bp and wild type (WT), while the GGTA1 genotypes of fetal pig F02 were -26 bp/+2 bp and -3 bp. The hCD55 mRNA expression levels of both fetal pigs were significantly higher than those of WT pigs (P＜0.01). The fetal pig F02 was selected as the donor cell source for recloning, 11 surviving piglets were obtained, all identified as GTKO/hCD55 gene-edited pigs. These pigs showed absence of α-Gal antigen expression, but weak or no expression of hCD55 was observed. Methylation analysis of the hCD55 gene's CpG island showed hypermethylation in kidney tissue lacking hCD55 expression, whereas it was not methylated or partially methylated in kidney tissue expressing hCD55. Moreover, codon optimization of the CpG island of the hCD55 gene to reduce CG content could achieve stable expression of the hCD55 gene. In addition, antigen-antibody binding experiment showed that the amount of human IgM binding to GTKO/hCD55 gene-edited pig fibroblasts was significantly lower than that of WT pigs (P＜0.01). Complement-dependent cytotoxicity experiment showed that the survival rate of fibroblasts in GTKO/hCD55 pigs was significantly higher than that in WT pigs (P＜0.01). Conclusion This study demonstrates the successful generation of GTKO/hCD55 gene-edited xenotransplant donor pigs. Methylation-induced gene silencing of the hCD55 gene can be effectively avoided by reducing the CG content of the CpG island through codon optimization. This study provides a reference for the development of xenotransplant donor pigs and guides subsequent research on xenotransplantation.

Key words: Xenotransplantation, Diannan miniature pigs, α-1,3-galactosyltransferase, Human CD55

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, 2025, 45(4): 379-392. 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, 2025, 45(4): 379-392. 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的筛选注：A，GGTA1基因敲除sgRNA设计和hCD55基因过表达载体的构建示意图；B，阳性单细胞35#和45#的GGTA1基因sgRNA靶区域的基因型；C，48个细胞单克隆hCD55基因的PCR鉴定电泳图；PC，阳性对照；NC，阴性对照；-，敲除碱基；+，插入碱基；M，DNA分子量标准；WT，野生型；WT （2/8）表示TA克隆后8个单菌落测序中有4种基因型包括2个WT、3个-134 bp、2个-26/+2 bp和1个-3 bp。

Figure 1 Screening of single-cell clones positive for GTKO/hCD55 gene editingNote： A， Design of sgRNA for GGTA1 gene knockout and schematic diagram of the vector for hCD55 gene overexpression； B， Genotype of the sgRNA targeted region of GGTA1 in positive single-cell clones 35# and 45#； C， PCR identification of the hCD55 gene from 48 single-cell clones； PC， Positive controls； NC， Negative controls； -， Indicates the knockout bases； +， Indicates the bases insertion； M， DNA ladder； WT， Wild type； WT （2/8） indicates among 8 single colonies sequencing after TA cloning， four genotypes were identified： two WT， three -134 bp， two -26/+2 bp and one -3 bp.

表1 胚胎移植和克隆仔猪的产生

Table 1 Embryo transfer and production of cloned piglets

序号 No.	仔猪编号 Piglet ID	仔猪出生体重/g Body weight/g	仔猪出生时状态 Status at birth	存活时间/d Survival time/d	代孕母猪编号 Recipients sow ID
1	DN-GTKO-hCD55F02P01	500	健康	3	P516
2	DN-GTKO-hCD55F02P02	400	健康	1	P516
3	DN-GTKO-hCD55F02P03	720	死胎	/	P452
4	DN-GTKO-hCD55F02P04	380	死胎	/
5	DN-GTKO-hCD55F02P05	620	腭裂	4
6	DN-GTKO-hCD55F02P06	200	木乃伊胎	/	P511
7	DN-GTKO-hCD55F02P07	560	死胎	/
8	DN-GTKO-hCD55F02P08	300	木乃伊胎	/
9	DN-GTKO-hCD55F02P09	400	木乃伊胎	/
10	DN-GTKO-hCD55F02P10	480	死胎	/	P443
11	DN-GTKO-hCD55F02P11	560	健康	2
12	DN-GTKO-hCD55F02P12	580	健康	3
13	DN-GTKO-hCD55F02P13	520	健康	1
14	DN-GTKO-hCD55F02P14	260	虚弱	2
15	DN-GTKO-hCD55F02P15	320	死胎	/
16	DN-GTKO-hCD55F02P16	620	健康	2
17	DN-GTKO-hCD55F02P17	740	健康	1
18	DN-GTKO-hCD55F02P18	560	死胎	/	P525
19	DN-GTKO-hCD55F02P19	540	健康	21
20	DN-GTKO-hCD55F02P20	660	健康	206

图2 GTKO/hCD55基因编辑克隆胎猪的鉴定注：A，2个胎龄33 d的克隆胎猪；B，胎猪心脏组织的性别鉴定PCR结果；C，胎猪心脏组织的血型基因PCR鉴定结果；D，胎猪心脏组织的hCD55基因的PCR鉴定；E，胎猪心脏组织的GGTA1基因sgRNA靶区域的PCR结果；F，胎猪心脏组织的GGTA1基因sgRNA靶区域的基因型；G，F01和F02胎猪心脏组织hCD55的mRNA表达水平；H，F02胎猪成纤维细胞中α-Gal和hCD55表达的流式细胞术分析结果。M，DNA分子量标准；WT，野生型；PC，阳性对照；NC，阴性对照；♂，雄性；♀，雌性；AO，AO型血；AA，AA型血；OO，OO型血；Blank，未加抗体的对照；F02，胎儿编号；??P＜0.01。

Figure 2 Identification of GTKO/hCD55 gene-edited cloned fetal pigsNote： A， Two cloned fetal pigs at 33 days of gestation； B，PCR results of sex identification of fetal pig heart tissue； C， PCR results of blood group gene identification of fetal pig heart tissue； D， PCR identification of hCD55 gene in fetal pig heart tissue； E， PCR results of the target region of GGTA1 gene sgRNA in fetal pig heart tissue； F， Genotype results of the target region of GGTA1 gene sgRNA in fetal pig heart tissue； G， hCD55 mRNA expression levels in fetal pig F01 and F02 heart tissues； H， Flow cytometric analysis results of α-Gal and hCD55 expression in fetal porcine F02 fibroblasts. M， DNA ladder； WT， Wild type； PC， Positive controls； NC， Negative controls； ♂， Male； ♀， Female； AO， AO blood type； AA， AA blood type； OO， OO blood type； Blank， Control without antibody； F02， Fetal number； ??P＜0.01.

图3 GTKO/hCD55基因编辑仔猪的鉴定注：A，GTKO/hCD55基因编辑克隆仔猪的照片；B，11头克隆仔猪hCD55基因的PCR鉴定结果；C，仔猪GGTA1基因sgRNA靶区域的PCR结果；D，仔猪P20 GGTA1基因sgRNA靶区域的基因型；E，仔猪P20 成纤维细胞中α-Gal和hCD55表达的流式细胞分析结果；F，仔猪肾脏组织的免疫荧光染色结果，标尺为50 μm。M，DNA分子量标准；WT，野生型。

Figure 3 Identification of GTKO/hCD55 gene-edited cloned pigletsNote： A， Photo of cloned piglets with GTKO/hCD55 gene editing； B， PCR identification results of hCD55 gene in 11 piglets； C， PCR results of the sgRNA target region of the piglet GGTA1 gene； D， GGTA1 genotype of the sgRNA target region in piglet P20； E， Flow cytometry analysis results of α-Gal and hCD55 expression in P20 piglet-derived fibroblasts； F， Immunofluorescence results of piglet kidney tissues，the scale bar indicates 50 μm. M， DNA ladder； WT， wild type.

图4 单细胞克隆混合体、胎猪、克隆仔猪中hCD55基因甲基化水平注：A，hCD55基因上的CpG岛示意图；B，C35&C45单细胞克隆混合体、克隆胎猪F02成纤维细胞、克隆仔猪（P01、P13、P12、P14、P17）肾脏组织中的hCD55基因甲基化水平。实心黑圆圈表示甲基化位点，空心圆圈表示无甲基化位点。

Figure 4 Methylation levels of hCD55 gene in mixed single-cell clones， fetal pigs， and cloned pigletsNote： A， Schematic representation of CpG island in the hCD55 gene； B， Methylation levels of hCD55 gene in mixed single-cell clones of C35&C45， cloned fetal pig F02 fibroblasts and cloned piglets （P01， P13， P12， P14， P17） kidney tissues. Solid black circles indicate methylation sites， and hollow circles indicate unmethylated sites.

图5 CpG岛区域密码子优化前后成纤维细胞中的hCD55表达水平注：A，密码子优化前后hCD55基因CpG岛区域的Sanger测序结果；B，优化前后（分别用hCD55CG 81%和hCD55CG 69%表示）载体分别转染猪成纤维细胞，流式细胞术检测hCD55在细胞中的表达水平（3次重复实验结果）。Blank，未加抗体的对照；WT，野生型。

Figure 5 Expression levels of hCD55 in fibroblasts before and after optimization of CpG island region codonNote： A， Sanger sequencing results of CpG island region in hCD55 gene before and after codon optimization； B， Porcine fibroblasts were transfected with the codon unoptimized and codon optimized vectors （represents by hCD55CG 81% and hCD55CG 69% respectively）， and the expression level of hCD55 in the cells was detected by flow cytometry （triplicate experimental results）. Blank， control without antibody； WT， wild type.

图6 GTKO/hCD55基因编辑猪的抗免疫排斥效果评价注：A，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分析；B，混合人血清孵育猪成纤维细胞后，补体对猪细胞的杀伤分析；C，图A与图B的统计分析结果。WT，野生型；GTKO/hCD55，实验组；Blank，未加抗体的对照；MFI，通过流式细胞仪检测到的荧光信号强度的平均值；CDC，补体依赖的细胞毒性实验；??P＜0.01；nsP>0.05。

Figure 6 Evaluation of anti-immune rejection efficacy of GTKO/hCD55 gene-edited pigsNote： A， Binding extent analysis of human IgG and IgM antibodies to porcine fibroblasts after incubation with mixed human serum； B， Porcine cell death analysis induced by complement after incubation of porcine fibroblasts with mixed human serum； C， The statistical analysis results of figures A and B. WT， wild-type； GTKO/hCD55， experimental group； Blank， control without antibody； MFI， mean fluorescence intensity； CDC， complement-dependent cytotoxicity； ??P＜0.01； nsP>0.05.

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GTKO/hCD55基因编辑异种器官移植供体猪的构建及功能验证

Construction and Functional Validation of GTKO/hCD55 Gene-Edited Xenotransplant Donor Pigs

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