A chromosome-scale reference genome of the Banna miniature inbred pig.

The Banna miniature inbred pig (BN) is an intensively inbred line for biomedical research and xenotransplantation due to its low individual variation and stable genetic background. Although it is originated from the Diannan miniature pig (DN), substantial genetic changes have actually occurred. However, the lack of a BN reference genome has limited studies on the complete genomic architecture and utilization as a biomedical model.

Generation of Knock-In Pigs Carrying Oct4-tdTomato Reporter through CRISPR/Cas9-Mediated Genome Engineering.

The porcine pluripotent cells that can generate germline chimeras have not been developed. The Oct4 promoter-based fluorescent reporter system, which can be used to monitor pluripotency, is an important tool to generate authentic porcine pluripotent cells. In this study, we established a porcine Oct4 reporter system, wherein the endogenous Oct4 promoter directly controls red fluorescent protein (RFP).

Generation of CRISPR/Cas9-mediated gene-targeted pigs via somatic cell nuclear transfer.

The domestic pig has been widely used as an important large animal model. Precise and efficient genetic modification in pig provides a great promise in biomedical research. Recently, clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system has been successfully used to produce many gene-targeted animals.

Highly efficient generation of GGTA1 biallelic knockout inbred mini-pigs with TALENs.

Inbred mini-pigs are ideal organ donors for future human xenotransplantations because of their clear genetic background, high homozygosity, and high inbreeding endurance. In this study, we chose fibroblast cells from a highly inbred pig line called Banna mini-pig inbred line (BMI) as donor nuclei for nuclear transfer, combining with transcription activator-like effector nucleases (TALENs) and successfully generated α-1,3-galactosyltransferase (GGTA1) gene biallelic knockout (KO) pigs. To validate the efficiency of TALEN vectors, in vitro-transcribed TALEN mRNAs were microinjected into one-cell stage parthenogenetically activated porcine embryos.

Comparison of the efficiency of Banna miniature inbred pig somatic cell nuclear transfer among different donor cells.

Somatic cell nuclear transfer (SCNT) is an important method of breeding quality varieties, expanding groups, and preserving endangered species. However, the viability of SCNT embryos is poor, and the cloned rate of animal production is low in pig. This study aims to investigate the gene function and establish a disease model of Banna miniature inbred pig.

[Cloning of splicing variants of alpha1,3-galactosyltransferase cDNA of Chinese Banna Minipig inbred line and its expression in human cells].

Objective: To study the transfection and expression of the splicing variants of alpha1, 3-galactosyltransferase cDNA of Chinese Banna Minipig Inbred Line (BMI) in human A549 cells.

Methods: Full length of alpha1,3-GT gene cDNA was amplified by RT-PCR from total RNA of BMI liver tissue and cloned into T-A cloning vector. Two different splicing variants of BMI alpha1,3-GT cDNA were confirmed by sequencing 15 positive clones and inserted respectively into pEGFP-N1 to construct eukaryotic expression vectors pN-GT1 and pN-GT2.

[Cloning of Chinese Banna minipig inbred-line alpha1,3-galactosyltransferase gene and construction of its recombinant eukaryotic expression vector].

This study sought to clone Chinese Banna minipig inbred-line (BMI) alpha1,3-galactosyltransferase (alpha1,3-GT) gene and construct its recombinant eukaryotic expression vector. Total RNA was isolated from BMI liver. Full length cDNA of alpha1,3-GT gene was amplified by RT-PCR and cloned into pMD18-T vector to sequence.

Expressed sequence tags analysis of a liver tissue cDNA library from a highly inbred minipig line.

Background: Porcine liver performing efficient physiological functions in the human body is prerequisite for successful liver xenotransplantation. However, the protein differences between pig and human remain largely unexplored. Therefore, we investigated the liver expression profile of a highly inbred minipig line.

Full-length cDNA cloning and protein three-dimensional structure modeling of porcine prothrombin.

Prothrombin is a vitamin K-dependent serine protease and plays pivotal roles in both procoagulant and anticoagulant pathway of hemostasis. In this study, we cloned the full-length cDNA of porcine prothrombin by cDNA library screening and SMART RACE technique. The full-length cDNA is 2027 bp, with a 1869 bp Open Reading Frame (ORF) coding 623 amino acids.

[Quantitative study on copy numbers of porcine endogenous retroviruses in genome of Banna miniature swine inbred line].

Objective: To investigate the copy numbers of porcine endogenous retroviruses in genome of Banna miniature swines for screening of donors in xenotransplantation with porcine transplants.

Methods: The genomic DNA of peripheral blood mononuclear cells(PBMCs) of 50 Banna miniature swines in 4 inbred sublines and 5 European Large White Pigs was extracted for the detection to pol, envA, envB, envC and envA/C recombinant in PERVs with real-time quantitative/semi-quantitative PCR and normal PCR.

Results: PCR products of pol, envA and envB of PERV could be obtained from all the Banna samples, and the mean amount of these products was 24.

[Antigen measurement and biomechanical characteristics of the inbred-line Banna mini-pig acellular bone matrix].

In this study, we prepared the acellular bone matrix of the inbred-line Banna mini-pig by using tissue engineering method and evaluated its possible application in bone tissue engineering. Histological analysis, xenoantigen expression and biomechanical measurement were performed on the matrix. HE staining and scanning electron microscopy showed the cellular components were almost removed.

Variation of host cell tropism of porcine endogenous retroviruses expressed in chinese Banna minipig inbred.

A serious donor-organ shortage urges the use of animal donors to treat a wide appropriate variety of major health problems including organ failure and diabetes. However, the promise of clinical xenotransplantation is offset at the present time by the potential of a public health risk due to the cross-species transmission of pathogens from animal donors to human patients. In particular, the transmission of porcine endogenous retrovirus (PERV) is a major concern.

Comparison of hepatic coagulant, fibrinolytic, and anticoagulant functions between Banna Minipig Inbred line and humans.

Background: As an ideal candidate for xenotransplantation, the compatibility of physiological porcine organs with those of humans is an essential premise. In this study, we analyzed hepatic coagulant, fibrinolytic, and anticoagulant functions between Banna Minipig Inbreds (BMIs) and humans to evaluate such hepatic compatibility.

Methods: BMI factors II, V, VII, X, and XII were added to the corresponding factor-deficient human plasma to determine prothrombin times (PT) and activated partial thromboplastin times (APTT).

[Comparison of hepatic protein synthesis function between Banna Minipig Inbred Line and human].

Objective: This is a research aimed at comparing the hepatic protein synthesis function of Banna Minipig Inbred Line (BMI) with that of human so as to evaluate the possibility of porcine liver replacement of human counterpart.

Methods: The venous blood samples from BMI and volunteer participants were collected. The albumin and total protein in the separated sera were tested using Beckman delta CX7 autoanalyzer, and serum protein electrophoresis was performed.

Phylogenetic relationship of porcine endogenous retrovirus (PERV) in Chinese pigs with some type C retroviruses.

PCR amplification of proviral DNA extracted from peripheral blood lymphocytes of three Chinese pigs (Banna minipig inbreed (BMI), Wu-Zhi-Shan pig (WZSP) and Neijiang pig (NJP)), using primers corresponding to highly conserved regions of reverse transcriptase (RT) of pol gene and nucleocapsid sequence of gag gene. PCR products were then extracted and cloned into pGEM-T vector. Phylogenetic analysis of the nucleotide sequences of PERV-BMI, PERV-WZSP and PERV-WZSP revealed that they were of retroviral origin.

[The quantity analysis of reverse transcriptase in porcine endogenous retrovirus expressed in Banna minipig inbred].

Quantitative RT(reverse transcriptase) assay was established to detect the reverse transcriptase in plasma of thirty-four Chinese Banna minipig inbred in this work. The protocol was given in the RT kit (Roche), using HIV-1 as the positive control of the kit and supernatant of PK-15 as the PERV positive control respectively. The results show that positive reverse transcriptase reaction can be detected in the plasma of the pigs, but the levels are much lower than that of HIV-1 and lower than that of PERV in supernatant of PK-15.