Production of Cloned Miniature Pigs Expressing High Levels of Human Apolipoprotein(a) in Plasma.

High lipoprotein(a) [Lp(a)] levels are a major risk factor for the development of atherosclerosis. However, because apolipoprotein(a) [apo(a)], the unique component of Lp(a), is found only in primates and humans, the study of human Lp(a) has been hampered due to the lack of appropriate animal models. Using somatic cell nuclear transfer (SCNT) techniques, we produced transgenic miniature pigs expressing human apo(a) in the plasma.

Effect of postactivation treatment with latrunculin A on in vitro and in vivo development of cloned embryos derived from kidney fibroblasts of an aged Clawn miniature boar.

The objective of this study was to examine the effect of postactivation treatment with latrunculin A (LatA), an actin polymerization inhibitor, on in vitro and in vivo development of somatic cell nuclear transfer (SCNT) embryos derived from kidney fibroblasts of an aged Clawn miniature boar (12 years old). After electric activation, SCNT embryos were treated with 0, 0.5 or 1 μM LatA and cultured in vitro.

Gene expression profile differences in embryos derived from prepubertal and adult Japanese Black cattle during in vitro development.

The present study was carried out to compare the gene expression profiles of in vitro-generated embryos derived from adult and prepubertal Japanese Black cattle oocytes using GeneChip Bovine Genome Array (containing 24072 probe sets representing over 23000 transcripts). Microarray experiments were performed on populations of 8- to 16-cell stage embryos and blastocysts derived from adult (24-35 months old) versus prepubertal (9-10 months old) Japanese Black cattle oocytes matured and fertilised in vitro. In total, 591 (2.

Expression analysis of an α-1, 3-galactosyltransferase, an enzyme that creates xenotransplantation-related α-Gal epitope, in pig preimplantation embryos.

α-1,3-Galactosyltransferase (α-GalT), an enzyme creating Galα1-3Gal (α-Gal) epitope on the cell surface in some mammalian species such as pigs, is known to be a key factor that causes hyperacute rejection upon transplantation from pigs to humans. To establish the RNA interference-based suppression of endogenous α-GalT messenger RNA (mRNA) synthesis in porcine preimplantation embryos, we determined the suitable embryonic stage at which stage such approach is possible by using the semi-quantitative RT-PCR (qRT-PCR) and the cytochemical method using a fluorescence-labeled Bandeiraea simplicifolia Isolectin B(4) (BS-I-B(4) ). Staining with BS-I-B(4) demonstrated that α-Gal epitope expression was first recognized at the 8-cell stage, and increased up to the hatched blastocyst stage.

Successful suppression of endogenous α-1,3-galactosyltransferase expression by RNA interference in pig embryos generated in vitro.

RNA interference (RNAi) technology using small interfering RNAs (siRNA) has been widely used as a powerful tool to knock down gene expression in various organisms. In pig preimplantation embryos, no attempt to suppress the target gene expression with such technology has been made. The purpose of this study is to demonstrate that the RNAi technology is useful for suppression of endogenous target gene expression at an early stage of development in pigs.

Whole-genome amplification-based GenomiPhi for multiple genomic analysis of individual early porcine embryos.

The multiple displacement amplification (MDA) method, which relies on isothermal DNA amplification using the DNA polymerase of the bacteriophage phi29, was recently developed for high-performance, whole-genome amplification (WGA). The objective of the present study was to determine whether a target sequence could be successfully amplified by conventional PCR when the genomic DNA of a single Day-7 porcine blastocyst (derived from SCNT of a gene-engineered fibroblast) was amplified by the MDA method and used as a template. The yield of double-stranded DNA was 103.

Effects of trichostatin A on in vitro development and transgene function in somatic cell nuclear transfer embryos derived from transgenic Clawn miniature pig cells.

The present study was carried out to examine the effects of post-activation treatment of trichostatin A (TSA), a histone deacetylase inhibitor, on in vitro development and transgene function of somatic cell nuclear transfer (SCNT) embryos derived from Clawn miniature pig embryonic fibroblast (PEF) transfected with a bacterial endo-β-galactosidase C gene (removal of the α-galactosyl (Gal) epitope). SCNT embryos were incubated with or without TSA (50 or 100 nmol/L) after activation, cultured in vitro and assessed for cleavage, blastocyst formation and transgene function. The rate of blastocyst formation was significantly higher in SCNT embryos treated with 50 nmol/L TSA than that in control (P < 0.

Production of genetically modified porcine blastocysts by somatic cell nuclear transfer: preliminary results toward production of xenograft-competent miniature pigs.

Galα1-3Gal (α-Gal epitope) is the major xenoantigenic epitope responsible for hyperacute rejection upon pig-to-human xenotransplantation. Endo-β-galactosidase C (EndoGalC) from Clostridium perfringens can digest the α-Gal epitope. In this study, gene-engineered primary cultured porcine embryonic fibroblasts (PEF) expressing EndoGalC were obtained and subjected to somatic cell nuclear transfer (SCNT) to test whether xenograft-competent pigs can be created.

Latrunculin A dramatically improves the developmental capacity of nuclear transfer embryos derived from gene-modified clawn miniature pig cells.

This study was carried out to examine the effect of postactivation treatment with latrunculin A (LatA), an actin polymerisation inhibitor, on in vitro and in vivo development of somatic cell nuclear transfer (SCNT) embryos derived from gene-modified Clawn miniature pig cells. After the fusion and activation, SCNT embryos were treated with or without a cytoskeletal inhibitor [LatA or 10.4 microM cytochalasin B (CB) for 2 h].

Stage-specific effects of osmolarity of a culture medium on development of pig oocytes and miniature pig somatic cell nuclear transfer embryos activated by ultrasound treatment.

Whether high osmolarity of a culture medium at the early culture stage affects the development of pig oocytes and miniature pig somatic cell nuclear transfer (SCNT) embryos activated by ultrasound was examined. When oocytes were cultured in modified porcine zygote medium-3 (mPZM-3) with increased NaCl to 138 mmol/L (mPZM-3+NaCl; 326 mOsm) or 50 mmol/L sucrose (mPZM-3+sucrose; 318 mOsm) for the first 2 days and then cultured in normal mPZM-3 (273 mOsm) for 5 days, the cleavage and blastocyst formation rates were significantly (P < 0.05) higher than those of oocytes cultured in mPZM-3 for 7 days.

Usefulness of a non-invasive reporter system for monitoring reprogramming state in pig cells: results of a cell fusion experiment.

Dedifferentiation of differentiated cells such as fibroblasts into pluripotent stem cells, so-called iPS cells, was first reported by Yamanaka et al., who successfully employed retroviral gene delivery of four stem-cell-specific transcription factors (Oct-3/4, Klf4, Sox2 and c-myc). Despite the mouse system in which an Oct-3/4 or Nanog promoter-based reporter system has already been established, there is no useful system in pigs for reporting the reprogramming state of gene-engineered cells.

Enrichment of xenograft-competent genetically modified pig cells using a targeted toxin, isolectin BS-I-B4 conjugate.

Background: The recent availability of alpha-1,3-galatosyltransferase knockout pigs has eliminated anti-Gal antibodies to the galalpha1-3gal (alphagal epitope) as the major barrier to xenotransplantation. These alphagal epitope-negative animals can also be produced by somatic cell nuclear transfer of cells overexpressing endo-beta-galactosidase (EndoGalC), an enzyme capable of digesting the alphagal epitope. For this, selection of cells with highly reduced synthesis of alphagal epitope is a prerequisite.

Valproic acid enhances in vitro development and Oct-3/4 expression of miniature pig somatic cell nuclear transfer embryos.

The present study was carried out to examine the effects of valproic acid (VPA), a histone deacetylase inhibitor, on in vitro development of miniature pig somatic cell nuclear transfer (SCNT) embryos and on expression of a mouse Oct-3/4 promoter-driven enhanced green fluorescent protein (EGFP) gene (EGFP expression only detected in Oct-3/4-expressing cells) introduced into donor cells for SCNT during their development. The addition of 4 mM VPA to embryo culture medium for 48 h after activation significantly (p < 0.01) increased the blastocyst formation rate of SCNT embryos compared with the control, whereas VPA did not affect their cleavage rate.

Enhancement of cytoplasmic maturation of in vitro-matured pig oocytes by mechanical vibration.

The effects of mechanical vibration during in vitro maturation and/or in vitro culture after artificial activation of pig oocytes on maturation and development were examined. In addition, the optimal conditions were applied to in vitro production of blastocysts derived from miniature pig somatic cell nuclear transfer (SCNT) embryos. Mechanical vibration during in vitro maturation did not affect the rates (60.

Beneficial effects of reversine on in vitro development of miniature pig somatic cell nuclear transfer embryos.

Reversine, a 2-(4-morpholinoanilino)-6-cyclohexylaminopurine analog, can induce dedifferentiation of myogenic lineage-committed cells into multipotent mesenchymal progenitor cells, from which osteoblasts and adipocytes redifferentiate under lineage-specific inducing conditions. Although the molecular mechanism of how reversine causes dedifferentiation of a differentiated cell has not been fully elucidated, we speculated that it would be involved in reprogramming. In the present study, we examined whether reversine can enhance the development of somatic cell nuclear transfer (SCNT) embryos by improving the reprogramming state of the somatic cell nuclei.

Development of a noninvasive monitoring system for evaluation of Oct-3/4 promoter status in miniature pig somatic cell nuclear transfer embryos.

The present study was carried out to develop a noninvasive monitoring system for evaluation of Oct-3/4 promoter gene status in miniature pig somatic cell nuclear transfer (SCNT) embryos during in vitro development. Miniature pig fetal fibroblasts (MPFFs) were transfected with a gene construct consisting of two expression units, a mouse Oct-3/4 promoter-driven enhanced green fluorescent protein (EGFP) gene (EGFP expression only detected in Oct-3/4-expressing cells) and a neomycin resistance gene. After neomycin selection, MPFFs that did not express EGFP were fused with enucleated pig oocytes, cultured in vitro and assessed for EGFP expression.

Effects of cycloheximide on parthenogenetic development of pig oocytes activated by ultrasound treatment.

The present study was carried out to examine the parthenogenetic development of pig oocytes treated with different concentrations of cycloheximide for different durations following activation by ultrasound stimulation. When oocytes were treated with 10 microg/ml cycloheximide for different durations, the blastocyst formation rate of oocytes treated for 5 h was significantly (P<0.05) higher than those of oocytes treated for 0-2 h.

Effects of demecolcine and sucrose on the incidence of cytoplasmic protrusions containing chromosomes in pig oocytes matured in vitro.

The present study was carried out to examine whether demecolcine and sucrose affect the formation of a cytoplasmic protrusion containing chromosomes in pig oocytes independently or in combination. In the presence of 20 mM sucrose, the rates of oocytes with a cytoplasmic protrusion after culture for 60 min with 0.2-1.

Efficient transfection of primarily cultured porcine embryonic fibroblasts using the Amaxa Nucleofection system.

Porcine embryonic fibroblasts (PEF) are important as donor cells for nuclear transfer for generation of genetically modified pigs. In this study, we determined an optimal protocol for transfection of PEF with the Amaxa Nucleofection system, which directly transfers DNA into the nucleus of cells, and compared its efficiency with conventional lipofection and electroporation. Cell survival and transfection efficiency were assessed using dye-exclusion assay and a green fluorescent protein (GFP) reporter construct, respectively.

Activation and parthenogenetic development of pig oocytes exposed to ultrasound in media containing different concentrations of Ca2+.

The present study was carried out to examine the activation and parthenogenetic development of pig oocytes after exposure to ultrasound in sorbitol media supplemented with different concentrations of Ca2+. The activation rates (68.8-75.

Birth of cloned miniature pigs derived from somatic cell nuclear transferred embryos activated by ultrasound treatment.

The present study was carried out to determine (1) the optimal duty cycle of ultrasound for activation of pig oocytes and cloned embryos derived from miniature pig fetal fibroblasts and (2) whether cloned embryos can develop to term following activation by ultrasound stimulation. When oocytes were exposed to ultrasound with 20% or 30% duty cycle, the blastocyst formation rates were significantly (P < 0.05) higher than that of oocytes exposed to ultrasound with 10% duty cycle.

In vitro development of cloned embryos derived from miniature pig somatic cells after activation by ultrasound stimulation.

The present study was carried out to examine the activation and development of cloned embryos produced by transferring miniature pig somatic cells into enucleated farm pig oocytes after exposing to ultrasound. The rates of the pronucleus-like structure formation and polar body-like structure extrusion in embryos exposed to ultrasound did not differ from those applied electric pulses. Although there was no significant difference in the blastocyst formation rates between different activation methods, the mean number of cells in the blastocysts developed from embryos activated by exposing to ultrasound was significantly (p < 0.

Optimization of Ca2+ concentrations in fusion and activation media for production of cloned embryos from miniature pig somatic cells.

The effects of Ca(2+) concentration in activation medium and cytochalasin B treatment after activation on the parthenogenetic development of pig oocytes were examined. In addition, cloned embryos derived from miniature pig somatic cells were activated under optimal conditions and the effects of Ca(2+) in fusion medium on the development of embryos after activation was examined. When oocytes were activated in 0.

Utility of ultrasound stimulation for activation of pig oocytes matured in vitro.

The present study was carried out to examine the development of pig oocytes after exposing to ultrasound under various conditions. When oocytes were exposed to ultrasound in the sorbitol medium, the blastocyst formation rate was significantly (P < 0.01) higher than that of oocytes exposed in HEPES-TLP-PVA.