Cloning of Macaque Monkeys by Somatic Cell Nuclear Transfer.

Generation of genetically uniform non-human primates may help to establish animal models for primate biology and biomedical research. In this study, we have successfully cloned cynomolgus monkeys (Macaca fascicularis) by somatic cell nuclear transfer (SCNT). We found that injection of H3K9me3 demethylase Kdm4d mRNA and treatment with histone deacetylase inhibitor trichostatin A at one-cell stage following SCNT greatly improved blastocyst development and pregnancy rate of transplanted SCNT embryos in surrogate monkeys. For SCNT using fetal monkey fibroblasts, 6 pregnancies were confirmed in 21 surrogates and yielded 2 healthy babies. For SCNT using adult monkey cumulus cells, 22 pregnancies were confirmed in 42 surrogates and yielded 2 babies that were short-lived. In both cases, genetic analyses confirmed that the nuclear DNA and mitochondria DNA of the monkey offspring originated from the nucleus donor cell and the oocyte donor monkey, respectively. Thus, cloning macaque monkeys by SCNT is feasible using fetal fibroblasts.

Generation and application of immortalized sheep fetal fibroblast cell line.

BACKGROUND: Primary sheep fetal fibroblasts (SFFCs) have emerged as a valuable resource for investigating the molecular and pathogenic mechanisms of orf viruses (ORFV). However, their utilization is considerably restricted due to the exorbitant expenses associated with their isolation and culture, their abbreviated lifespan, and the laborious procedure. RESULTS: In our investigation, the primary SFFCs were obtained and immortalized by introducing a lentiviral recombinant plasmid containing the large T antigen from simian virus 40 (SV40). The expression of fibronectin and vimentin proteins, activity of SV40 large T antigen, cell proliferation assays, and analysis of programmed cell death revealed that the immortalized large T antigen SFFCs (TSFFCs) maintained the same physiological characteristics and biological functions as the primary SFFCs. Moreover, TSFFCs demonstrated robust resistance to apoptosis, extended lifespan, and enhanced proliferative activity compared to primary SFFCs. Notably, the primary SFFCs did not undergo in vitro transformation or exhibit any indications of malignancy in nude mice. Furthermore, the immortalized TSFFCs displayed live ORFV vaccine susceptibility. CONCLUSIONS: Immortalized TSFFCs present valuable in vitro models for exploring the characteristics of ORFV using various techniques. This indicates their potential for secure utilization in future studies involving virus isolation, vaccine development, and drug screening.

Efficient delivery of a large-size Cas9-EGFP vector in porcine fetal fibroblasts using a Lonza 4D-Nucleofector system.

BACKGROUND: Porcine fetal fibroblasts (PFFs) are important donor cells for generating genetically modified pigs, but the transfection efficiencies of PFFs are often unsatisfactory especially when large-size vectors are to be delivered. In this study, we aimed to optimize the transfection conditions for delivery of a large-size vector in PFFs using Lonza 4D-Nucleofector™ vessels and strips. METHODS: We firstly delivered a 13 kb Cas9-EGFP and a 3.5 kb pMAX-GFP vector into PFFs via 7 programs recommended by the Lonza basic protocol. We then tested 6 customized dual-electroporation programs for delivering the 13 kb plasmid into PFFs. In addition, we screened potential alternative electroporation buffers to the Nucleofector™ P3 solution. Finally, three CRISPR/Cas9-sgRNAs targeting Rosa26, H11, and Cep112 loci were delivered into PFFs with different single and dual-electroporation programs. RESULTS: Notably lower transfection efficiencies were observed when delivering the 13 kb vector than delivering the 3.5 kb vector in PFFs via the single-electroporation programs. The customized dual-electroporation program FF-113 + CA-137 exhibited higher transfection efficiencies than any of the single-electroporation programs using vessels (98.1%) or strips (89.1%) with acceptable survival rates for the 13 kb vector. Entranster-E buffer generated similar transfection efficiencies and 24-hour survival rates to those from the P3 solution, thus can be used as an alternative electroporation buffer. In the genome-editing experiments, the FF-113 + CA-137 and CA-137 + CA-137 programs showed significantly superior (P < 0.01) efficiencies to ones from the single-electroporation programs in vessels and strips. Entranster-E buffer produced higher indel efficiencies than the P3 buffer. CONCLUSIONS: We markedly increased the delivery efficiencies for a large vector via customized dual-electroporation programs using Lonza 4D-Nucleofector™ system, and Entranster-E buffer can be used as an alternative electroporation buffer to Nucleofector™ P3 buffer.

Cell therapy for full-thickness wounds: are fetal dermal cells a potential source?

The application of autologous dermal fibroblasts has been shown to improve burn wound healing. However, a major hurdle is the availability of sufficient healthy skin as a cell source. We investigated fetal dermal cells as an alternative source for cell-based therapy for skin regeneration. Human (hFF), porcine fetal (pFF) or autologous dermal fibroblasts (AF) were seeded in a collagen-elastin substitute (Novomaix, NVM), which was applied in combination with an autologous split thickness skin graft (STSG) to evaluate the effects of these cells on wound healing in a porcine excisional wound model. Transplantation of wounds with NVM+hFF showed an increased influx of inflammatory cells (e.g., neutrophils, macrophages, CD4(+) and CD8(+) lymphocytes) compared to STSG, acellular NVM (Acell-NVM) and NVM+AF at post-surgery days 7 and/or 14. Wounds treated with NVM+pFF presented only an increase in CD8(+) lymphocyte influx. Furthermore, reduced alpha-smooth muscle actin (αSMA) expression in wound areas and reduced contraction of the wounds was observed with NVM+AF compared to Acell-NVM. Xenogeneic transplantation of NVM+hFF increased αSMA expression in wounds compared to NVM+AF. An improved scar quality was observed for wounds treated with NVM+AF compared to Acell-NVM, NVM+hFF and NVM+pFF at day 56. In conclusion, application of autologous fibroblasts improved the overall outcome of wound healing in comparison to fetal dermal cells and Acell-NVM, whereas application of fetal dermal fibroblasts in NVM did not improve wound healing of full-thickness wounds in a porcine model. Although human fetal dermal cells demonstrated an increased immune response, this did not seem to affect scar quality.

Effect of Co-transfection of Anti-myostatin shRNA Constructs in Caprine Fetal Fibroblast Cells.

Knockdown of myostatin gene (MSTN), transforming growth factor-ß superfamily, and a negative regulator of the skeletal muscle growth, by RNA interference (RNAi), has been reported to increase muscle mass in mammals. The current study was aimed to cotransfect two anti-MSTN short hairpin RNA (shRNA) constructs in caprine fetal fibroblast cells for transient silencing of MSTN gene. In the present investigation, approximately 89% MSTN silencing was achieved in transiently transfected caprine fetal fibroblast cells by cotransfection of two best out of four anti-MSTN shRNA constructs. Simultaneously, we also monitored the induction of IFN responsive genes (IFN), pro-apoptotic gene (caspase3) and anti-apoptotic gene (MCL-1) due to cotransfection of different anti-MSTN shRNA constructs. We observed induction of 0.66-19.12, 1.04-4.14, 0.50-3.43, and 0.42-1.98 for folds IFN-ß, OAS1, caspase3, and MCL-1 genes, respectively (p < 0.05). This RNAi based cotransfection method could provide an alternative strategy of gene knockout and develop stable caprine fetal fibroblast cells. Furthermore, these stable cells can be used as a cell donor for the development of transgenic cloned embryos by somatic cell nuclear transfer (SCNT) technique.

Analysis of cell growth and gene expression of porcine adipose tissue-derived mesenchymal stem cells as nuclear donor cell.

In several laboratory animals and humans, adipose tissue-derived mesenchymal stem cells (ASC) are of considerable interest because they are easy to harvest and can generate a huge proliferation of cells from a small quantity of fat. In this study, we investigated: (i) the expression patterns of reprogramming-related genes in porcine ASC; and (ii) whether ASC can be a suitable donor cell type for generating cloned pigs. For these experiments, ASC, adult skin fibroblasts (AF) and fetal fibroblasts (FF) were derived from a 4-year-old female miniature pig. The ASC expressed cell-surface markers characteristic of stem cells, and underwent in vitro differentiation when exposed to specific differentiation-inducing conditions. Expression of DNA methyltransferase (DNMT)1 in ASC was similar to that in AF, but the highest expression of the DNMT3B gene was observed in ASC. The expression of OCT4 was significantly higher in FF and ASC than in AF (P < 0.05), and SOX2 showed significantly higher expression in ASC than in the other two cell types (P < 0.05). After somatic cell nuclear transfer (SCNT), the development rate of cloned embryos derived from ASC was comparable to the development of those derived using FF. Total cell numbers of blastocysts derived using ASC and FF were significantly higher than in embryos made with AF. The results demonstrated that ASC used for SCNT have a potential comparable to those of AF and FF in terms of embryo in vitro development and blastocyst formation.

Cloning and Characterization of Yak DHODH Gene and Its Functional Studies in a Bisphenol S-Induced Ferroptosis Model of Fetal Fibroblasts.

Dihydroorotate dehydrogenase (DHODH) is a rate-limiting enzyme of de novo biosynthesis of pyrimidine. Although the involvement of DHODH in resisting ferroptosis has been successively reported in recent years, which greatly advanced the understanding of the mechanism of programmed cell death (PCD), the genetic sequence of the yak DHODH gene and its roles in ferroptosis are still unknown. For this purpose, we firstly cloned the coding region sequence of DHODH (1188 bp) from yak liver and conducted a characterization analysis of its predictive protein that consists of 395 amino acids. We found that the coding region of the yak DHODH gene presented high conservation among species. Second, the expression profile of the DHODH gene in various yak tissues was investigated using RT-qPCR. The results demonstrated that DHODH was widely expressed in different yak tissues, with particularly high levels in the spleen, heart, and liver. Third, to investigate the involvement of DHODH in regulating ferroptosis in cells, yak skin fibroblasts (YSFs) were isolated from fetuses. And then, bisphenol S (BPS) was used to induce the in vitro ferroptosis model of YSFs. We observed that BPS decreased the cell viability (CCK8) and membrane potential (JC-1) of YSFs in a dose-dependent manner and induced oxidative stress by elevating reactive oxygen species (ROS). Simultaneously, it was evident that BPS effectively augmented the indicators associated with ferroptosis (MDA and BODIPY staining) and reduced GSH levels. Importantly, the co-administration of Ferrostatin-1 (Fer), a potent inhibitor of ferroptosis, significantly alleviated the aforementioned markers, thereby confirming the successful induction of ferroptosis in YSFs by BPS. Finally, overexpression plasmids and siRNAs of the yak DHODH gene were designed and transfected respectively into BPS-cultured YSFs to modulate DHODH expression. The findings revealed that DHODH overexpression alleviated the occurrence of BPS-induced ferroptosis, while interference of DHODH intensified the ferroptosis process in YSFs. In summary, we successfully cloned the coding region of the yak DHODH gene, demonstrating its remarkable conservation across species. Moreover, using BPS-induced ferroptosis in YSFs as the model, the study confirmed the role of the DHODH gene in resisting ferroptosis in yaks. These results offer valuable theoretical foundations for future investigations into the functionality of the yak DHODH gene and the underlying mechanisms of ferroptosis in this species.

MDM2 antagonists promote CRISPR/Cas9-mediated precise genome editing in sheep primary cells.

CRISPR-Cas9-mediated genome editing in sheep is of great use in both agricultural and biomedical applications. While targeted gene knockout by CRISPR-Cas9 through non-homologous end joining (NHEJ) has worked efficiently, the knockin efficiency via homology-directed repair (HDR) remains lower, which severely hampers the application of precise genome editing in sheep. Here, in sheep fetal fibroblasts (SFFs), we optimized several key parameters that affect HDR, including homology arm (HA) length and the amount of double-stranded DNA (dsDNA) repair template; we also observed synchronization of SFFs in G2/M phase could increase HDR efficiency. Besides, we identified three potent small molecules, RITA, Nutlin3, and CTX1, inhibitors of p53-MDM2 interaction, that caused activation of the p53 pathway, resulting in distinct G2/M cell-cycle arrest in response to DNA damage and improved CRISPR-Cas9-mediated HDR efficiency by 1.43- to 4.28-fold in SFFs. Furthermore, we demonstrated that genetic knockout of p53 could inhibit HDR in SFFs by suppressing the expression of several key factors involved in the HDR pathway, such as BRCA1 and RAD51. Overall, this study offers an optimized strategy for the usage of dsDNA repair template, more importantly, the application of MDM2 antagonists provides a simple and efficient strategy to promote CRISPR/Cas9-mediated precise genome editing in sheep primary cells.

Vitamin C down-regulates the H3K9me3-dependent heterochromatin in buffalo fibroblasts via PI3K/PDK1/SGK1/KDM4A signal axis.

The success of reprogramming is dependent on the reprogramming factors enriched in the cytoplasm of recipient oocytes and the potential of donor nucleus to be reprogrammed. Histone 3 lysine 9 trimethylation (H3K9me3) was identified as a major epigenetic barrier impeding complete reprogramming. Treating donor cell with vitamin C (Vc) can enhance the developmental potential of cloned embryos, but the underlying mechanisms still need to be elucidated. In this study, we found that 20µg/mL Vc could promote proliferation and inhibit apoptosis of BFFs, as well as down-regulate the H3K9me3-dependent heterochromatin and increase chromatin accessibility. Inhibited the expression of KDM4A resulted in increasing apoptosis rate and the H3K9me3-dependent heterochromatin, which can be restored by Vc. Moreover, Vc up-regulated the expression of KDM4A through PI3K/PDK1/SGK1 pathway. Inhibiting any factor in the signal axis of this PI3K pathway not only suppressed the activity of KDM4A but also substantially increased the level of H3K9me3 modification and the expression of the HP1α protein. Finally, Vc can rescue those negative effects induced by the blocking the PI3K/PDK1/SGK1 pathway. Collectively, Vc can down-regulate the H3K9me3-dependent heterochromatin in BFFs via PI3K/PDK1/SGK1/KDM4A signal axis, suggesting that Vc can turn the chromatin status of donor cells to be reprogrammed more easily.

Pig Cloning Using Somatic Cell Nuclear Transfer.

Porcine cloning technology can be used to produce progenies genetically identical to the donor cells from high-quality breeding pigs. In addition, genetically modified pigs have been produced by somatic cell nuclear transfer using genetically modified porcine fetal fibroblasts. The method of preparing genetically modified pigs is critical for establishing pig models for human diseases, and for generating donor animals for future xenotransplantation. This chapter describes detailed procedures for generating cloned pigs using fetal fibroblasts as nuclear donors.

Generation of Induced Dopaminergic Neurons from Human Fetal Fibroblasts.

Since the first demonstration of direct dopaminergic neuronal reprogramming, over a dozen methods have been developed to generate induced dopaminergic neurons from various sources of cells. Here, we first present an overview of the different methods to generate induced neurons of a generic type and of different subtypes, with a particular focus on induced dopaminergic neurons generated from human fibroblasts. We then describe a protocol to generate induced dopaminergic neurons from commercially available human fetal lung fibroblasts. These cells could serve for various biomedical application, including regenerative medicine for conditions such as Parkinson's disease.

MicroRNA expression data of pluripotent and somatic cells and identification of cell type-specific MicroRNAs in pigs.

Typical models of pluripotency, humans and mice, have been used to analyse the characteristics of pluripotent stem cells. However, these species exhibit molecular differences in many aspects. With similar physiology and genomics as humans, pigs are promising model for the research of pluripotency. The data of porcine pluripotent cells would be helpful in understanding the molecular network of human pluripotency. Pluripotent cells of humans and mice exhibit specific MicroRNA (miRNA) expression patterns to maintain the pluripotent state. Information about miRNA expression in pig pluripotent cells is not sufficient, so we analysed miRNAs in pluripotent (blastocysts and ES-like) and somatic cell samples (PEB and PFF). We screened cell-type specific miRNAs and identified their target genes. Functional annotation of the target genes was also conducted. Our data may facilitate miRNA-based induction and maintenance of the pluripotent state of porcine cells and provide support to fill the gap between the pluripotency networks of humans and mice.

Impaired Replication Timing Promotes Tissue-Specific Expression of Common Fragile Sites.

Common fragile sites (CFSs) are particularly vulnerable regions of the genome that become visible as breaks, gaps, or constrictions on metaphase chromosomes when cells are under replicative stress. Impairment in DNA replication, late replication timing, enrichment of A/T nucleotides that tend to form secondary structures, the paucity of active or inducible replication origins, the generation of R-loops, and the collision between replication and transcription machineries on particularly long genes are some of the reported characteristics of CFSs that may contribute to their tissue-specific fragility. Here, we validated the induction of two CFSs previously found in the human fetal lung fibroblast line, Medical Research Council cell strain 5 (MRC-5), in another cell line derived from the same fetal tissue, Institute for Medical Research-90 cells (IMR-90). After induction of CFSs through aphidicolin, we confirmed the expression of the CFS 1p31.1 on chromosome 1 and CFS 3q13.3 on chromosome 3 in both fetal lines. Interestingly, these sites were found to not be fragile in lymphocytes, suggesting a role for epigenetic or transcriptional programs for this tissue specificity. Both these sites contained late-replicating genes NEGR1 (neuronal growth regulator 1) at 1p31.1 and LSAMP (limbic system-associated membrane protein) at 3q13.3, which are much longer, 0.880 and 1.4 Mb, respectively, than the average gene length. Given the established connection between long genes and CFS, we compiled information from the literature on all previously identified CFSs expressed in fibroblasts and lymphocytes in response to aphidicolin, including the size of the genes contained in each fragile region. Our comprehensive analysis confirmed that the genes found within CFSs are longer than the average human gene; interestingly, the two longest genes in the human genome are found within CFSs: Contactin Associated Protein 2 gene (CNTNAP2) in a lymphocytes' CFS, and Duchenne muscular dystrophy gene (DMD) in a CFS expressed in both lymphocytes and fibroblasts. This indicates that the presence of very long genes is a unifying feature of all CFSs. We also obtained replication profiles of the 1p31.1 and 3q13.3 sites under both perturbed and unperturbed conditions using a combination of fluorescent in situ hybridization (FISH) and immunofluorescence against bromodeoxyuridine (BrdU) on interphase nuclei. Our analysis of the replication dynamics of these CFSs showed that, compared to lymphocytes where these regions are non-fragile, fibroblasts display incomplete replication of the fragile alleles, even in the absence of exogenous replication stress. Our data point to the existence of intrinsic features, in addition to the presence of long genes, which affect DNA replication of the CFSs in fibroblasts, thus promoting chromosomal instability in a tissue-specific manner.

Improved Delivery of CRISPR/Cas9 System Using Magnetic Nanoparticles into Porcine Fibroblast.

Genetically modified pigs play an important role in agriculture and biomedical research; hence, new efficient methods are needed to obtain genetically engineered cells and animals. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas (CRISPR-associated) system represents an effective genome editing tool. It consists of two key molecules: single guide RNA (sgRNA) and the Cas9 endonuclease that can be introduced into the cells as one plasmid. Typical delivery methods for CRISPR/Cas9 components are limited by low transfection efficiency or toxic effects on cells. Here, we describe the use of magnetic nanoparticles and gradient magnetic field to improve delivery of CRISPR/Cas9 constructs into porcine fetal fibroblasts. Polyethylenimine-coated nanoparticles with magnetic iron oxide core were used to form magnetic plasmid DNA lipoplexes. CRISPR/Cas9 construct was prepared to induce site-specific cutting at the porcine H11 locus. Quantitative assessment of genomic cleavage by sequence trace decomposition demonstrated that the magnetofection efficiency was more than 3.5 times higher compared to the classic lipofection method. The Tracking of Indels by Decomposition web tool precisely determined the spectrum of indels that occurred. Simultaneously, no additional cytotoxicity associated with the utilization of magnetic nanoparticles was observed. Our results indicate that magnetofection enables effective delivery of the CRISPR/Cas9 construct into porcine fetal fibroblasts with low cell toxicity.

Porcine Fetal-Derived Fibroblasts Alter Gene Expression and Mitochondria to Compensate for Hypoxic Stress During Culture.

The Warburg effect is characterized by decreased mitochondrial oxidative phosphorylation and increased glycolytic flux in adequate oxygen. The preimplantation embryo has been described to have characteristics of the Warburg effect, including similar changes in gene expression and mitochondria, which are more rudimentary in appearance. We hypothesized hypoxia would facilitate anaerobic glycolysis in fibroblasts thereby promoting gene expression and media metabolite production reflecting the Warburg effect hallmarks in early embryos. Additionally, we speculated that hypoxia would induce a rudimentary small mitochondrial phenotype observed in several cell types evidenced to demonstrate the Warburg effect. While many have examined the role hypoxia plays in pathological conditions, few studies have investigated changes in primary cells which could be used in somatic cell nuclear transfer. We found that cells grown in 1.25% O2 had normal cell viability and more, but smaller mitochondria. Several hypoxia-inducible genes were identified, including seven genes for glycolytic enzymes. In conditioned media from hypoxic cells, the quantities of gluconolactone, cytosine, and uric acid were decreased indicating higher consumption than control cells. These results indicate that fibroblasts alter gene expression and mitochondria to compensate for hypoxic stress and maintain viability. Furthermore, the metabolic changes observed, making them more similar to preimplantation embryos, could be facilitating nuclear reprogramming making these cells more amendable to future use in somatic cell nuclear transfer.

Suppressing Ku70/Ku80 expression elevates homology-directed repair efficiency in primary fibroblasts.

The main DNA repair pathways, nonhomologous end joining (NHEJ) and homology-directed repair (HDR), are complementary to each other; hence, interruptions of the NHEJ pathway can favor HDR. Improving HDR efficiency in animal primary fibroblasts can facilitate the generation of gene knock-in animals with agricultural and biomedical values by somatic cell nuclear transfer. In this study, we used siRNA to suppress the expression of Ku70 and Ku80, which are the key factors in NHEJ pathway, to investigate the effect of Ku silencing on the HDR efficiency in pig fetal fibroblasts. Down-regulation of Ku70 and Ku80 resulted in the promotion of the frequencies of multiple HDR pathways, including homologous recombination, single strand annealing, and single-stranded oligonucleotide-mediated DNA repair. We further evaluated the effects of Ku70 and Ku80 silencing on promoting HR-mediated knock-in efficiency in two porcine endogenous genes and found a significant increase in the amount of knock-in cells in Ku-silenced fibroblasts compared with control. The RNA interference strategy will benefit the generation of cell lines and organisms with precise genetic modifications.

Efficient Knock-in of a Point Mutation in Porcine Fibroblasts Using the CRISPR/Cas9-GMNN Fusion Gene.

During CRISPR/Cas9 mediated genome editing, site-specific double strand breaks are introduced and repaired either unspecific by non-homologous end joining (NHEJ) or sequence dependent by homology directed repair (HDR). Whereas NHEJ-based generation of gene knock-out is widely performed, the HDR-based knock-in of specific mutations remains a bottleneck. Especially in primary cell lines that are essential for the generation of cell culture and animal models of inherited human diseases, knock-in efficacy is insufficient and needs significant improvement. Here, we tested two different approaches to increase the knock-in frequency of a specific point mutation into the MYH7-gene in porcine fetal fibroblasts. We added a small molecule inhibitor of NHEJ, SCR7 (5,6-bis((E)-benzylideneamino)-2-mercaptopyrimidin-4-ol), during genome editing and screened cell cultures for the point mutation. However, this approach did not yield increased knock-in rates. In an alternative approach, we fused humanized Cas9 (hCas9) to the N-terminal peptide of the Geminin gene (GMNN). The fusion protein is degraded in NHEJ-dominated cell cycle phases, which should increase HDR-rates. Using hCas9-GMNN and point mutation-specific real time PCR screening, we found a two-fold increase in genome edited cell cultures. This increase of HDR by hCas9-GMNN provides a promising way to enrich specific knock-in in porcine fibroblast cultures for somatic cloning approaches.

Establishment of PCR Identification Method for Pig Blood Type / 实验动物与比较医学

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.

Differential developmental competence and gene expression patterns in buffalo (Bubalus bubalis) nuclear transfer embryos reconstructed with fetal fibroblasts and amnion mesenchymal stem cells.

The developmental ability and gene expression pattern at 8- to 16-cell and blastocyst stages of buffalo (Bubalus bubalis) nuclear transfer (NT) embryos from fetal fibroblasts (FFs), amnion mesenchymal stem cells (AMSCs) and in vitro fertilized (IVF) embryos were compared in the present studies. The in vitro expanded buffalo FFs showed a typical "S" shape growth curve with a doubling time of 41.4 h and stained positive for vimentin. The in vitro cultured undifferentiated AMSCs showed a doubling time of 39.5 h and stained positive for alkaline phosphatase, and these cells also showed expression of pluripotency markers (OCT 4, SOX 2, NANOG), and mesenchymal stem cell markers (CD29, CD44) and were negative for haematopoietic marker (CD34) genes at different passages. Further, when AMSCs were exposed to corresponding induction conditions, these cells differentiated into adipogenic, chondrogenic and osteogenic lineages which were confirmed through oil red O, alcian blue and alizarin staining, respectively. Donor cells at 3-4 passage were employed for NT. The cleavage rate was significantly (P < 0.05) higher in IVF than in FF-NT and AMSC-NT embryos (82.6 ± 8.2 vs. 64.6 ± 1.3 and 72.3 ± 2.2 %, respectively). However, blastocyst rates in IVF and AMSC-NT embryos (30.6 ± 2.7 and 28.9 ± 3.1 %) did not differ and were significantly (P < 0.05) higher than FF-NT (19.5 ± 1.8 %). Total cell number did not show significant (P > 0.05) differences between IVF and AMSC-NT embryos (186.7 ± 4.2, 171.2 ± 3.8, respectively) but were significantly (P < 0.05) higher than that from FF-NT (151.3 ± 4.1). Alterations in the expression pattern of genes implicated in transcription and pluripotency (OCT4, STAT3, NANOG), DNA methylation (DNMT1, DNMT3A), histone deacetylation (HDAC2), growth factor signaling and imprinting (IGF2, IGF2R), apoptosis (BAX, BCL2), metabolism (GLUT1) and oxidative stress (MnSOD) regulation were observed in cloned embryos. The transcripts or expression patterns in AMSC-NT embryos more closely followed that of the in vitro derived embryos compared with FF-NT embryos. The results demonstrate that multipotent amnion MSCs have a greater potential as donor cells than FFs in achieving enhanced production of cloned buffalo embryos.