Whole Blood Transcriptome Analysis for Lifelong Monitoring in Elite Sniffer Dogs Produced by Somatic Cell Nuclear Transfer.

Reproductive cloning by somatic cell nuclear transfer (SCNT) is a valuable method to propagate service dogs with desirable traits because of higher selection rates in cloned dogs. However, incomplete reprogramming is a major barrier to SCNT, and the assessment of reprogramming is limited to preimplantation embryos and tissues from dead and/or adult tissue. Thus, lifelong monitoring in SCNT dogs can be useful to evaluate the SCNT service dogs for propagation.

Stable Regulation of Senescence-Related Genes in Galactose-alpha1,3-galactose Epitope Knockout and Human Membrane Cofactor Protein hCD46 Pig.

Background: Pigs are considered suitable animal donor models for xenotransplantation. For successful organ transplantation, immune rejection must be overcome. Xenotransplantation has recently been successfully performed using galactose-alpha1,3-galactose epitopes knockout (GalTKO) and a human membrane cofactor protein (hCD46) in a pig model.

Immune molecular profiling of whole blood drawn from a non-human primate cardiac xenograft model treated with anti-CD154 monoclonal antibodies.

Most studies of xenografts have been carried out with complex immunosuppressive regimens to prevent immune rejection; however, such treatments may be fatal owing to unknown causes. Here, we performed immune molecular profiling following anti-CD154 monoclonal antibody (mAb) treatment in heterotopic abdominal cardiac xenografts from α-1,3-galactosyltransferase-knockout pigs into cynomolgus monkeys to elucidate the mechanisms mediating the undesirable fatal side effects of immunosuppressive agents. Blood samples were collected from healthy monkeys as control and then at 2 days after xenograft transplantation and just before humane euthanasia; 94 genes related to the immune system were analyzed.

Scriptaid improves the reprogramming of donor cells and enhances canine-porcine interspecies embryo development.

Histone methylation, histone acetylation, and DNA methylation are the important factors for somatic cell nuclear transfer (SCNT). Histone deacetylase inhibitors (HDACi) and DNA methyltransferase inhibitors (DNMTi) have been used to improve cloning efficiency. In particular, scriptaid, an HDACi, has been shown to improve SCNT efficiency.

Production and Breeding of Transgenic Cloned Pigs Expressing Human CD73.

One of the reasons to causing blood coagulation in the tissue of xenografted organs was known to incompatibility of the blood coagulation and anti-coagulation regulatory system between TG pigs and primates. Thus, overexpression of human CD73 (hCD73) in the pig endothelial cells is considered as a method to reduce coagulopathy after pig-to-non-human-primate xenotransplantation. This study was performed to produce and breed transgenic pigs expressing hCD73 for the studies immune rejection responses and could provide a successful application of xenotransplantation.

Production of transgenic pig as an Alzheimer's disease model using a multi-cistronic vector system.

Alzheimer's disease (AD) is a progressive neurodegenerative disease associated with memory loss and cognitive impairments. An AD transgenic (Tg) pig model would be useful for preclinical testing of therapeutic agents. We generated an AD Tg pig by somatic cell nuclear transfer (SCNT) using a multi-cistronic vector that harbored three AD-related genes with a total of six well-characterized mutations: hAPP (K670N/M671L, I716V, and V717I), hTau (P301L), and hPS1 (M146V and L286P).

Effects of Cell Cycle Regulators on the Cell Cycle Synchronization of Porcine induced Pluripotent Stem Cells.

Unlike mouse results, cloning efficiency of nuclear transfer from porcine induced pluripotent stem cells (piPSCs) is very low. The present study was performed to investigate the effect of cell cycle inhibitors on the cell cycle synchronization of piPSCs. piPSCs were generated using combination of six human transcriptional factors under stem cell culture condition.

Dog cloning with in vivo matured oocytes obtained using electric chemiluminescence immunoassay-predicted ovulation method.

Radioactive immunoassay (RIA) is a traditional serum hormone assay method, but the application of the method in reproductive studies is limited by the associated radioactivity. The aim of present study was to evaluate the reliability of RIA and to compare its canine serum progesterone concentration determination accuracy to that of the electric chemiluminescence immunoassay (ECLI). In vivo matured oocytes were utilized for canine somatic cell nuclear transfer (SCNT), and serum progesterone levels were assessed to accurately determine ovulation and oocyte maturation.

Generation of α-1,3-galactosyltransferase knocked-out transgenic cloned pigs with knocked-in five human genes.

Recent progress in genetic manipulation of pigs designated for xenotransplantation ha6s shown considerable promise on xenograft survival in primates. However, genetic modification of multiple genes in donor pigs by knock-out and knock-in technologies, aiming to enhance immunological tolerance against transplanted organs in the recipients, has not been evaluated for health issues of donor pigs. We produced transgenic Massachusetts General Hospital piglets by knocking-out the α-1,3-galactosyltransferase (GT) gene and by simultaneously knocking-in an expression cassette containing five different human genes including, DAF, CD39, TFPI, C1 inhibitor (C1-INH), and TNFAIP3 (A20) [GT] that are connected by 2A peptide cleavage sequences to release individual proteins from a single translational product.

Molecular immunology profiles of monkeys following xenografting with the islets and heart of α-1,3-galactosyltransferase knockout pigs.

Effective immunosuppression strategies and genetically modified animals have been used to prevent hyperacute and acute xenograft rejection; however, the underlying mechanisms remain unknown. In this study, we evaluated the expression of a comprehensive set of immune system-related genes (89 genes, including five housekeeping genes) in the blood of cynomolgus monkeys (~5 yr old) used as graft recipients, before and after the xenografting of the islets and heart from single and double α-1,3-galactosyltransferase (GalT) knockout (KO) pigs (<6 weeks old). The immunosuppressive regimen included administration of cobra venom factor, anti-thymocyte globulin, rituximab, and anti-CD154 monoclonal antibodies to recipients before and after grafting.

Production of Cloned Korean Native Pig by Somatic Cell Nuclear Transfer.

The Korean native pig (KNP) have been considered as animal models for animal biotechnology research because of their relatively small body size and their presumably highly inbred status due to the closed breeding program. However, little is reported about the use of KNP for animal biotechnology researches. This study was performed to establish the somatic cell nuclear transfer (SCNT) protocol for the production of swine leukocyte antigens (SLA) homotype-defined SCNT KNP.

IMPROVED SURVIVAL AND DEVELOPMENTAL RATES IN VITRIFIED-WARMED PIG OOCYTES AFTER RECOVERY CULTURE WITH COENZYME Q10.

Background: The primary problems with porcine oocyte vitrification are their low viability and development; both need improvement.

Objective: This study was designed to improve the survival and developmental rates in vitrified-warmed porcine oocytes.

Materials And Methods: Porcine oocytes matured in vitro were vitrified-warmed with Cryotop.

HIGH INCIDENCE OF POLYSPERMIC FERTILIZATION IN BOVINE OOCYTES MATURED IN VITRO AFTER CRYOTOP VITRIFICATION.

Background: Vitrification with the Cryotop device is the most promising technique for oocyte cryopreservation, but the high post-warming morphological survival of bovine oocytes does not guarantee high developmental competence after in vitro fertilization (IVF).

Objective: This study was designed to examine achievement of normal fertilization in bovine oocytes vitrified-warmed with the Cryotop device.

Materials And Methods: Oocytes were matured in vitro and vitrified-warmed after complete removal of the cumulus layers.

Generation of leukemia inhibitory factor-dependent induced pluripotent stem cells from the Massachusetts General Hospital miniature pig.

The generation and application of porcine induced pluripotent stem cells (iPSCs) may enable the testing for safety and efficacy of therapy in the field of human regenerative medicine. Here, the generation of iPSCs from the Massachusetts General Hospital miniature pig (MGH minipig) established for organ transplantation studies is reported. Fibroblasts were isolated from the skin of the ear of a 10-day-old MGH minipig and transduced with a cocktail of six human factors: POU5F1, NANOG, SOX2, C-MYC, KLF4, and LIN28.

Nucleofection-mediated α1,3-galactosyltransferase gene inactivation and membrane cofactor protein expression for pig-to-primate xenotransplantation.

Xenotransplantation of pig organs into primates leads to hyperacute rejection (HAR). Functional ablation of the pig α 1,3-galactosyltransferase (GalT) gene, which abrogates expression of the Gal α 1-3Gal β 1-4GlcNAc-R (Gal) antigen, which inhibits HAR. However, antigens other than Gal may induce immunological rejection by their cognate antibody responses.

Promoter activity analysis and methylation characterization of LTR elements of PERVs in NIH miniature pig.

The potential risk of porcine endogenous retrovirus (PERV) transmission is an important issue in xenotransplantation (pig-to-human transplantation). Long terminal repeats (LTRs) in PERV elements show promoter activity that could affect neighboring functional genes. The methylation status and promoter activities of 3 LTR structures (PERV-LTR1, LTR2, and LTR3 elements) belonging to the PERV-A family were examined using luciferase reporter genes in human liver cell lines (HepG2 and Hep3B).

Improvement of cloning efficiency in minipigs using post-thawed donor cells treated with roscovitine.

Massachusetts General Hospital miniature pigs (MGH minipigs) have been established for organ transplantation studies across the homozygous major histocompatibility complex, but cloning efficiency of MGH minipigs is extremely low. This study was designed to increase the productivity of MGH minipigs by nuclear transfer of post-thaw donor cells after 1 h co-incubation with roscovitine. The MGH minipig cells were genetically modified with GT KO (alpha1,3-galactosyltransferase knock-out) and hCD46 KI (human CD46 knock-in) and used as donor cells.

Identification and promoter analysis of PERV LTR subtypes in NIH-miniature pig.

Porcine endogenous retroviruses (PERVs) are integrated into the genomes of all pigs. Since some PERVs can also infect human cells, they represent a potential risk for xenotransplantation involving pig cells or organs. The long terminal repeat (LTR) elements of PERVs show promoter activity that can affect human functional genes; therefore, we examined these elements in this study.

No long-term feeding toxicities on the health status in rats fed with cloned Korean native beef cattle (Hanwoo) meat.

This study was designed to undertake a risk assessment to identify the health status of rats fed with somatic cell nuclear transfer (SCNT)-cloned Korean native beef cattle (Hanwoo) meat for 26 weeks. The rats were randomly divided into 5 groups, each consisting of 12 male (142.6 ± 5.

Aberrant gene expression patterns in extraembryonic tissue from cloned porcine embryos.

The abnormal development of embryos reconstructed by somatic cell nuclear transfer (SCNT) is considered to be associated with consequent changes in gene expression following errors in epigenetic reprogramming. In this study, we carried out SCNT using donor fibroblast cells derived from 3-way hybrids (Landrace×Duroc×Yorkshire). A total of 655 SCNT embryos were transferred, and 6.

Phenotypic characterization of Hanwoo (native Korean cattle) cloned from somatic cells of a single adult.

We investigated phenotypic differences in Hanwoo cattle cloned from somatic cells of a single adult. Ten genetically identical Hanwoo were generated by somatic cell nuclear transfer from a single adult. Weights at birth, growing pattern, horn and noseprint patterns were characterized to investigate phenotypic differences.

In vitro and in vivo genotoxic effects of somatic cell nuclear transfer cloned cattle meat.

Although the nutritional composition and health status after consumption of the meat and milk derived from both conventionally bred (normal) and somatic cell nuclear transferred (cloned) animals and their progeny are not different, little is known about their food safeties like genetic toxicity. This study is performed to examine both in vitro (bacterial mutation and chromosome aberration) and in vivo (micronucleus) genotoxicity studies of cloned cattle meat. The concentrations of both normal and cloned cattle meat extracts (0-10×) were tested to five strains of bacteria (Salmonella typhimurium: TA98, TA100, TA1535, and TA1537; Escherichia coli: WP2uvrA) for bacterial mutation and to Chinese hamster lung (CHL/IU) cells for chromosome aberration, respectively.

A diet of somatic cell nuclear transfer cloned-cattle meat produced no toxic effects on behavioral or reproductive characteristics of F1 rats derived from dams fed on cloned-cattle meat.

Background: The composition and nutritional value of meat and milk derived from cloned animals and their progeny has not been demonstrated to be different from normal animals, but possible food consumption risks that might arise from unidentified hazards remain. In this study, we investigated the effects of somatic cell nuclear transfer cloned-cattle meat diet on the behavioral and reproductive characteristics of F1 rats derived from dams that were also fed on cloned-cattle meat.

Methods And Results: F1 rats were divided into five diet groups with their dams: commercial pellets (control), pellets containing 5% (N-5) and 10% (N-10) of normal-cattle meat, and diets containing 5% (C-5) and 10% (C-10) of cloned-cattle meat.

Trichostatin A promotes the development of bovine somatic cell nuclear transfer embryos.

We studied the effects of trichostatin A (TSA), a histone deacetylase inhibitor, on the development of bovine somatic cell nuclear transfer (SCNT) embryos by investigating (1) the optimal concentration and treatment time of TSA for development of bovine SCNT embryos, (2) the status of histone acetylation in TSA-treated and control SCNT embryos and (3) the expression of histone acetylation- and deacetylation-related genes in TSA-treated and control SCNT embryos. We observed that 50 nM TSA-treatment for 20 h following fusion resulted in more efficient in vitro development of bovine SCNT embryos to the blastocyst stage. In regard to histone H4K5 acetylation, half of the control SCNT embryos faintly displayed histone H4K5 signals 30 min after electrofusion, while most of the TSA-treated SCNT embryos displayed histone H4K5 signals within 30 min after electrofusion.