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/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

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. DOI: 10.12300/j.issn.1674-5817.2025.024.

Figures/Tables 7

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.

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

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.

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.

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.

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）.

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.

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