Establishment of PCR Identification Method for Pig Blood Type

doi:10.12300/j.issn.1674-5817.2023.065

Abstract

Abstract:

Objective Xenotransplantation is an effective way to address the shortage of human organ donors, but it faces serious immune rejection reactions, including hyperacute rejection caused by blood type differences. Establishing a stable, convenient, and reliable method for pig blood type identification can quickly screen suitable donor pigs for xenograft research. Methods Banna miniature inbred pigs, Diannan small eared pigs, and Bama Xiang pigs were selected as the research objects. DNA was extracted from the blood, oral buccal mucosa, and fetal fibroblasts of the three strains of pigs using DNA extraction kits. The target fragment of the ABO homologous gene EAA intron 7 in pigs was amplified using PCR method. Blood agglutination reaction was used to detect hemolysis in pig anterior vena cava whole blood after adding anti A and B antibodies. Immunohistochemical method was used to detect the expression level of A antigen in pig heart, liver, spleen, lung, and kidney tissues. Immunofluorescence method was used to detect the expression level of A antigen in pig oral mucosa. By comparing the results of different methods for determining pig blood types, the stability and reliability of the PCR method were verified, and a convenient PCR based pig blood type identification method was established. Results Firstly, the blood PCR results of 69 inbred strains of Banna miniature pigs, 7 Diannan small eared pigs, and 34 Bama Xiang pigs showed 20 AO blood types, 66 AA blood types, and 24 O blood types. The PCR results of fetal fibroblasts from 47 Diannan small eared pigs showed that all 47 fetuses were O blood type. Among them, the oral mucosal PCR results of 8 gene edited cloned pigs were consistent with those of donor fetal fibroblasts, all of which were O blood type. The oral mucosal PCR results of 8 wild-type pigs (2 inbred lines of Banna miniature pigs, 4 Diannan small eared pigs, and 2 Bama Xiang pigs) were consistent with the blood PCR identification results. Then, 11 inbred lines of Banna miniature pigs, 4 Diannan small eared pigs, and 2 Bama Xiang pigs were randomly selected for blood agglutination reaction validation, and the results were consistent with the PCR identification results of both blood samples and oral mucosa samples. Moreover, immuno-histochemical analysis was performed on the heart, liver, lung, kidney, and spleen tissues of one Banna miniature pig inbred line and two Bama Xiang pigs, and the results were consistent with blood PCR identification and blood agglutination reaction results. Finally, oral mucosal samples were collected from 2 inbred strains of Banna miniature pigs and 1 Bama Xiang pig for immunofluorescence detection, and the results were consistent with the blood PCR identification results. Conclusion By collecting fetal cells and oral mucosal samples from live pigs for PCR detection, the blood type of pigs can be accurately and efficiently identified, providing a convenient method for blood type screening of xenograft donor pigs.

Key words: Pigs, Blood type, PCR technology, Xenotransplantation, Blood, Fetal fibroblasts, Oral mucosa

CLC Number:

R-332

Jiaoxiang WANG, Yan WANG, Ke HU, Kaixiang XU, Taiyun WEI, Deling JIAO, Heng ZHAO, Hongye ZHAO, HongJiang WEI. Establishment of PCR Identification Method for Pig Blood Type[J]. Laboratory Animal and Comparative Medicine, 2023, 43(6): 585-594.

Figures/Tables 7

Figure 1 Schematic diagram of the PCR method for identifying pig blood types （differences in EAA gene between pig A and O blood types）

Figure 2 PCR identification results of blood types in different strains of pigsNote：A， The blood PCR identification results of 69 Banna miniature inbred pigs （BNP01-69）； B， The blood PCR identification results of 7 Diannan small eared pigs （DNP01-7）； C， The blood PCR identification results of 34 Bama Xiang pigs （BMP01-34）. M12000， M5000 and M2000 are DNA Markers. AOC， AAC， and OOC respectively refer to the AO， AA， and OO blood group standard control samples that have been sequenced in our laboratory in the previous stage.

Figure 3 PCR identification of blood types in fetal fibroblasts and oral mucosal cells of Diannan cloned small eared pigsNote：A， PCR identification results of 17 wild-type Diannan small eared pig fetal fibroblasts （DNF1-17）； B， PCR identification results of 18 porcine gene-editing fetuses fibroblasts of Diannan small eared pigs （DNF18-35）； C， PCR identification results of 12 porcine gene-editing fetuses fibroblasts of Diannan small eared pigs （DNF36-47）；D， PCR identification results of oral mucosal samples from 8 gene-editing cloned Diannan small eared pigs （DNP08-15）； E， PCR identification results of oral mucosal samples from 8 wild- type pigs （BNP11-12， Two Banna miniature inbred pigs； DNP01，03，04 and 06， four Diannan small eared pigs； BMP31-32， two Bama Xiang pigs）. M2000 is the DNA Marker. AOC， AAC， and OOC respectively refer to the AO， AA， and OO blood group standard control samples that have been sequenced in our laboratory in the previous stage.

Figure 4 Results of the blood agglutination reaction for identifying pig blood typesNote：A, Blood agglutination reaction of 11 Banna miniature inbred pigs (BNP02-08, 10-13); B, Blood agglutination reaction of Diannan small eared pigs (DNP03-06); C, Blood agglutination reaction of 2 Bama Xiang pigs (BMP31-32). AA, AO, and OO refer to the results of PCR identification of blood types, which were consistent with the results of blood agglutination reaction.

Figure 5 Results of immunohistochemical identification of pig blood types （DAB staining, all the bars are 100 μm）Note：BNP， Banna miniature inbred pigs； BMP， Bama Xiang pigs. AA， AO and OO refer to the results of blood type identification by PCR， which are consistent with the results of blood type identification by immunohistochemistry.

Figure 6 Results of immunofluorescence identification of pig blood types in oral mucosal samples (all the bars are 100 μm)Note： BNP06， 12 are two Banna miniature inbred pigs； BMP31 is one Bama Xiang pig. FITC, Fluorescein isothiocyanate； DAPI， 4'，6-diamidine-2-phenylindole. AA， AO and OO refer to the results of blood PCR identification of blood types， which are consistent with the results of immunofluorescence identification of blood types.

Table 1 Comparison of PCR, hemagglutination test, immunohistochemistry and immunofluorescence staining results

序号Serial number	编号Number	血液 PCR Blood PCR	血液凝集反应 Hemagglutination test		免疫组织化学 Immunohistochemistry					颊黏膜PCR PCR of buccal mucosal samples	免疫荧光 Immunofluorescence
序号Serial number	编号Number	血液 PCR Blood PCR	抗A抗体Anti-A antibody	抗B抗体Anti-B antibody	心 Heart	肝 Liver	脾 Spleen	肺 Lung	肾 Kidney	颊黏膜PCR PCR of buccal mucosal samples	免疫荧光 Immunofluorescence
1	BNP02	AA	+	-
2	BNP03	AA	+	-
3	BNP04	AA	+	-
4	BNP05	AA	+	-
5	BNP06	AO	+	-							A
6	BNP07	AA	+	-	-	A	A	A	A
7	BNP08	AA	+	-
8	BNP09	AA	+	-
9	BNP10	AA	+	-
10	BNP11	AA	+	-						AA
11	BNP12	AA	+	-						AA	A
12	DNP03	AO	+	-						AO
13	DNP04	OO	-	-						OO
14	DNP05	OO	-	-
15	DNP06	AO	+	-						AO
16	BMP02	OO			-	-	-	-	-
17	BMP24	AO			-	A	-	A	A
18	BMP31	OO	-	-						OO	-
19	BMP32	OO	-	-						OO

References 18

1	GRIFFITH B P, GOERLICH C E, SINGH A K, et al. Genetically modified Porcine-to-human cardiac xenotransplantation[J]. N Engl J Med, 2022, 387(1):35-44. DOI: 10.1056/NEJMoa2201422 .
2	NIU D, WEI H J, LIN L, et al. Inactivation of porcine endogenous retrovirus in pigs using CRISPR-Cas9[J]. Science, 2017, 357(6357):1303-1307. DOI: 10.1126/science.aan4187 .
3	YUE Y N, XU W H, KAN Y N, et al. Extensive germline genome engineering in pigs[J]. Nat Biomed Eng, 2021, 5(2):134-143. DOI: 10.1038/s41551-020-00613-9 .
4	RYDBERG L. ABO-incompatibility in solid organ transplantation[J]. Transfus Med, 2001, 11(4):325-342. DOI: 10.1046/j.1365-3148.2001.00313.x .
5	BUSCH J, SPECHT S, EZZELARAB M, et al. Buccal mucosal cell immunohistochemistry: a simple method of determining the ABH phenotype of baboons, monkeys, and pigs[J]. Xenotransplantation, 2006, 13(1):63-68. DOI: 10.1111/j.1399-3089.2005.00255.x .
6	PETAZZI P, MIQUEL-SERRA L, HUERTAS S, et al. ABO gene editing for the conversion of blood type A to universal type O in Rh_null donor-derived human-induced pluripotent stem cells[J]. Clin Transl Med, 2022, 12(10): e1063. DOI: 10.1002/ctm2.1063 .
7	SMITH D M, NEWHOUSE M, NAZIRUDDIN B, et al. Blood groups and transfusions in pigs[J]. Xenotransplantation, 2006, 13(3):186-194. DOI: 10.1111/j.1399-3089.2006.00299.x .
8	YAMAMOTO F, YAMAMOTO M. Molecular genetic basis of porcine histo-blood group AO system[J]. Blood, 2001, 97(10):3308-3310. DOI: 10.1182/blood.v97.10.3308 .
9	TAWEECHAIPAISANKUL A, KIM G A, JIN J X, et al. Establishment and identification of cell lines from type O blood Korean native pigs and their efficiency in supporting embryonic development via somatic cell nuclear transfer[J]. J Vet Sci, 2018, 19(4):492-499. DOI: 10.4142/jvs.2018.19.4.492 .
10	CHOI M K, LE M T, CHO H, et al. Determination of complete sequence information of the human ABO blood group orthologous gene in pigs and breed difference in blood type frequencies[J]. Gene, 2018, 640:1-5. DOI: 10.1016/j.gene. 2017. 09.047 .
11	SONG W Q, ZHOU S H, SHAO L N, et al. Non-invasive fetal ABO genotyping in maternal plasma using real-time PCR[J]. Clin Chem Lab Med, 2015, 53(12):1943-1950. DOI: 10.1515/cclm-2015-0011 .
12	HSU F H, SERPEDIN E, CHEN Y D, et al. Stochastic modeling of the relationship between copy number and gene expression based on transcriptional logic[J]. IEEE Trans Biomed Eng, 2012, 59(1):272-280. DOI: 10.1109/TBME. 2011. 2173341 .
13	HERNÁNDEZ-GÓMEZ C, HERNÁNDEZ-LEMUS E, ESPINAL-ENRÍQUEZ J. The role of copy number variants in gene Co-expression patterns for luminal B breast tumors[J]. Front Genet, 2022, 13:806607. DOI: 10.3389/fgene.2022.806607 .
14	ORIOL R, YE Y, KOREN E, et al. Carbohydrate antigens of pig tissues reacting with human natural antibodies as potential targets for hyperacute vascular rejection in pig-to-man organ xenotransplantation[J]. Transplantation, 1993, 56(6):1433-1442. DOI: 10.1097/00007890-199312000-00031 .
15	ZHAO H, LI Y Y, WIRIYAHDAMRONG T, et al. Improved production of GTKO/hCD55/hCD59 triple-gene-modified Diannan miniature pigs for xenotransplantation by recloning[J]. Transgenic Res, 2020, 29(3):369-379. DOI: 10.1007/s11248-020-00201-2 .
16	王娇祥, 范柠粼, 施德佳, 等. 通过微量细胞鉴定快速生产基因编辑猪[J]. 云南农业大学学报(自然科学), 2021, 36(5):811-819, 825. DOI: 10.12101/j.issn.1004-390X(n).202103095 .
	WANG J X, FAN N L, SHI D J, et al. Rapid production of gene-editing pigs by identifying minimum numbers of positive fibroblasts for SCNT[J]. J Yunnan Agric Univ, 2021, 36(5):811-819, 825. DOI: 10.12101/j.issn.1004-390X(n).202103095 .
17	ZENG R, ZENG Y Z. Molecular cloning and characterization of SLA-DR genes in the 133-family of the Banna mini-pig inbred line[J]. Animal Genet, 2005, 36(3):267-269. DOI: 10.1111/j.1365-2052.2005.01277.x .
18	WEI H J, QING Y B, PAN W R, et al. Comparison of the efficiency of Banna miniature inbred pig somatic cell nuclear transfer among different donor cells[J]. PLoS One, 2013, 8(2): e57728. DOI: 10.1371/journal.pone.0057728 .

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