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

doi:10.12300/j.issn.1674-5817.2025.024

Abstract

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

CLC Number:

Q95-33

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.

Figures/Tables 7

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.

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

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.

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.

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.

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.

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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