ObjectiveXenotransplantation 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.MethodsBanna 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 pigblood 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.ConclusionBy 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.

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.

Narcotic drugs and class Ⅰ psychotropic substances have always been special management drugs in medical institutions. Although relevant policies and regulations have been introduced at the national level ，there are problems of poor policy understanding and inconsistent implementation in medical institutions in the process of policy implementation. In order to standardize the management of narcotic drugs and class Ⅰ psychotropic substances in medical institutions of Yunnan province ， based on the preliminary research and Delphi expert consultation ，this editing group finally formed the Expert Consensus on Key Links Quality Control Management of Narcotic Drugs and Class I Psychotropic Substances in Medical Institutions of Yunnan Province by focusing on the key aspects of quality control of key links for narcotic drugs and class Ⅰ psychotropic substances in medical institutions ，aiming to provide reference for the clinical use and management of narcotic drugs and class Ⅰ psychotropic substances in medical institutions of Yunnan province.