Recent developments in genome editing technology using meganucleases demonstrate an efficient method of producing gene edited pigs. In this study, we examined the effectiveness of the transcription activator-like effector nuclease (TALEN) system in generating specific mutations on the pig genome. Specific TALEN was designed to induce a double-strand break on exon 9 of the porcine α1,3-galactosyltransferase (GGTA1) gene as it is the main cause of hyperacute rejection after xenotransplantation. Human decay-accelerating factor (hDAF) gene, which can produce a complement inhibitor to protect cells from complement attack after xenotransplantation, was also integrated into the genome simultaneously. Plasmids coding for the TALEN pair and hDAF gene were transfected into porcine cells by electroporation to disrupt the porcine GGTA1 gene and express hDAF. The transfected cells were then sorted using a biotin-labeled IB4 lectin attached to magnetic beads to obtain GGTA1 deficient cells. As a result, we established GGTA1 knockout (KO) cell lines with biallelic modification (35.0%) and GGTA1 KO cell lines expressing hDAF (13.0%). When these cells were used for somatic cell nuclear transfer, we successfully obtained live GGTA1 KO pigs expressing hDAF. Our results demonstrate that TALEN-mediated genome editing is efficient and can be successfully used to generate gene edited pigs.
Animals ; Antigens, CD55/genetics ; Cell Line ; DNA Breaks, Double-Stranded ; Exons/genetics ; Galactosyltransferases/*genetics ; Gene Editing/*veterinary ; Gene Knockout Techniques ; Humans ; Nuclear Transfer Techniques ; Swine ; Transcription Activator-Like Effector Nucleases/*genetics/*metabolism

Somatic cell nuclear transfer (SCNT) is a cost-effective technique for producing transgenic pigs. However, abnormalities in the cloned pigs might prevent use these animals for clinical applications or disease modeling. In the present study, we generated several cloned pigs. One of the pigs was found to have intrapancreatic ectopic splenic tissue during histopathology analysis although this animal was grossly normal and genetically identical to the other cloned pigs. Ectopic splenic tissue in the pancreas is very rare, especially in animals. To the best of our knowledge, this is the first such report for cloned pigs.
Animals ; Animals, Genetically Modified ; Choristoma/pathology/*veterinary ; Cloning, Organism ; Nuclear Transfer Techniques/*veterinary ; *Pancreas ; Splenic Diseases/pathology/*veterinary ; Swine ; Swine Diseases/*pathology ; Swine, Miniature

The objective of the present study was to investigate the effects of three different culture media on the development of canine somatic cell nuclear transfer (SCNT) embryos. Canine cloned embryos were cultured in modified synthetic oviductal fluid (mSOF), porcine zygote medium-3 (PZM-3), or G1/G2 sequential media. Our results showed that the G1/G2 media yielded significantly higher morula and blastocyst development in canine SCNT embryos (26.1% and 7.8%, respectively) compared to PZM-3 (8.5% and 0%) or mSOF (2.3% and 0%) media. In conclusion, this study suggests that blastocysts can be produced more efficiently using G1/G2 media to culture canine SCNT embryos.
Animals ; Blastocyst/cytology ; Cloning, Organism/*veterinary ; Culture Media/metabolism ; Dogs/*embryology ; Embryo Culture Techniques/*veterinary ; *Embryonic Development ; Nuclear Transfer Techniques/*veterinary

This study was conducted to investigate the effects of rapamycin treatment during in vitro maturation (IVM) on oocyte maturation and embryonic development after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) in pigs. Morphologically good (MGCOCs) and poor oocytes (MPCOCs) were untreated or treated with 1 nM rapamycin during 0-22 h, 22-42 h, or 0-42 h of IVM. Rapamycin had no significant effects on nuclear maturation and blastocyst formation after PA of MGCOCs. Blastocyst formation after PA was significantly increased by rapamycin treatment during 22-42 h and 0-42 h (46.6% and 46.5%, respectively) relative to the control (33.3%) and 0-22 h groups (38.6%) in MPCOCs. In SCNT, blastocyst formation tended to increase in MPCOCs treated with rapamycin during 0-42 h of IVM relative to untreated oocytes (20.3% vs. 14.3%, 0.05 < p < 0.1), while no improvement was observed in MGCOCs. Gene expression analysis revealed that transcript abundance of Beclin 1 and microtubule-associated protein 1 light chain 3 mRNAs was significantly increased in MPCOCs by rapamycin relative to the control. Our results demonstrated that autophagy induction by rapamycin during IVM improved developmental competence of oocytes derived from MPCOCs.
Animals ; Embryonic Development/*drug effects ; Female ; In Vitro Oocyte Maturation Techniques/veterinary ; Nuclear Transfer Techniques/*veterinary ; Oocytes/growth & development ; *Parthenogenesis ; Sirolimus/*pharmacology ; Sus scrofa/*growth & development/metabolism

This study was conducted to investigate the expression of three genes related to early embryonic development in bovine transgenic cloned embryos. To accomplish this, development of bovine transgenic somatic cell nuclear transfer (SCNT) embryos was compared with non-transgenic embryos. Next, mRNA transcription of three specific genes (DNMT1, Hsp 70.1, and Mash2) related to early embryo development in transgenic SCNT embryos was compared between transgenic and non-transgenic SCNTs, parthenogenetic embryos, and in vitro fertilization (IVF) embryos. Transgenic SCNT embryos showed significantly lower rates of development to the blastocyst stage than non-transgenic ones. To investigate normal gene expression, RNA was extracted from ten blastocysts derived from parthenogenesis, IVF, non-transgenic, and transgenic SCNT embryos and reverse-transcribed to synthesize cDNA. The cDNA was then subjected to PCR amplification and semi-quantified. More DNMT1 mRNA was detected in the transgenic SCNT group than the other three groups. Hsp 70.1 mRNA was detected in the IVF embryos, while lower levels were found in SCNT and parthenogenetic embryos. Mash2 mRNA was present at the highest levels in transgenic SCNT embryos. In conclusion, the higher levels of methylation and lower protein synthesis after heat shock in the transgenic SCNT embryos expected based on our results may cause lower embryonic development.
Animals ; Animals, Genetically Modified/genetics ; Basic Helix-Loop-Helix Transcription Factors/*genetics/metabolism ; Cattle/embryology/*genetics ; DNA (Cytosine-5-)-Methyltransferase/*genetics/metabolism ; Embryo, Mammalian/embryology/metabolism ; Female ; Fertilization in Vitro ; *Gene Expression Regulation, Developmental ; HSP70 Heat-Shock Proteins/*genetics/metabolism ; Nuclear Transfer Techniques/veterinary ; Parthenogenesis ; Pregnancy ; RNA, Messenger/genetics/metabolism ; Transcription, Genetic

The level of P4 at the time of embryo transfer (ET) is important. P4 concentrations and numbers of corpora lutea for 126 recipients were evaluated. Nuclear transfer embryos were transferred into 126 surrogates. 11 maintained their pregnancy until full-term delivery, 17 miscarried, and implantation failed in 98 animals. P4 levels in the full-term group were significantly different from those of the pigs that aborted or in which implantation failed (p < 0.05). However, the numbers of corpora lutea were not significantly different. These findings indicate that the concentration of progesterone can be an important factor for successful ET in pigs.
Animals ; Corpus Luteum/*physiology ; Embryo Transfer/*veterinary ; Embryo, Mammalian/*physiology ; Female ; Nuclear Transfer Techniques ; Pregnancy ; *Pregnancy Rate ; Progesterone/*blood ; Retrospective Studies ; Sus scrofa/*physiology

Various somatic cell nuclear transfer (SCNT) techniques for mammalian species have been developed to adjust species-specific procedures to oocyte-associated differences among species. Species-specific SCNT protocols may result in different expression levels of developmentally important genes that may affect embryonic development and pregnancy. In the present study, porcine oocytes were treated with demecolcine that facilitated enucleation with protruding genetic material. Enucleation and donor cell injection were performed either simultaneously with a single pipette (simplified one-step SCNT; SONT) or separately with different pipettes (conventional two-step SCNT; CTNT) as the control procedure. After blastocysts from both groups were cultured in vitro, the expression levels of developmentally important genes (OCT4, NANOG, EOMES, CDX2, GLUT-1, PolyA, and HSP70) were analyzed by real-time quantitative polymerase chain reaction. Both the developmental rate according to blastocyst stage as well as the expression levels CDX2, EOMES, and HSP70 were elevated with SONT compared to CTNT. The genes with elevated expression are known to influence trophectoderm formation and heat stress-induced arrest. These results showed that our SONT technique improved the development of SCNT porcine embryos, and increased the expression of genes that are important for placental formation and stress-induced arrest.
Animals ; Biological Markers/metabolism ; Cloning, Organism ; Embryo, Mammalian/metabolism ; Female ; *Gene Expression Regulation, Developmental ; Nuclear Transfer Techniques/instrumentation/*veterinary ; Oocytes/metabolism ; Pregnancy ; Real-Time Polymerase Chain Reaction ; Swine/*embryology/*genetics

The generation of reactive oxygen species (ROS) and subsequent mitochondrial and DNA damage in bovine somatic cell nuclear transfer (SCNT) embryos were examined. Bovine enucleated oocytes were electrofused with donor cells and then activated by a combination of Ca-ionophore and 6-dimethylaminopurine culture. The H2O2 and .OH radical levels, mitochondrial morphology and membrane potential (DeltaPsi), and DNA fragmentation of SCNT and in vitro fertilized (IVF) embryos at the zygote stage were analyzed. The H2O2 (35.6 +/- 1.1 pixels/embryo) and .OH radical levels (44.6 +/- 1.2 pixels/embryo) of SCNT embryos were significantly higher than those of IVF embryos (19.2 +/- 1.5 and 23.8 +/- 1.8 pixels/embryo, respectively, p < 0.05). The mitochondria morphology of SCNT embryos was diffused within the cytoplasm. The DeltaPsi of SCNT embryos was significantly lower (p < 0.05) than that of IVF embryos (0.95 +/- 0.04 vs. 1.21 +/- 0.06, red/green). Moreover, the comet tail length of SCNT embryos was longer than that of IVF embryos (515.5 +/- 26.4 microm vs. 425.6 +/- 25.0 microm, p < 0.05). These results indicate that mitochondrial and DNA damage increased in bovine SCNT embryos, which may have been induced by increased ROS levels.
Animals ; *Apoptosis ; Caspase 3/metabolism ; Cattle ; Colorimetry/veterinary ; Comet Assay/veterinary ; *DNA Damage ; DNA, Mitochondrial/*genetics/metabolism ; Embryo Transfer/veterinary ; Embryo, Mammalian/*cytology/embryology ; Fertilization in Vitro/veterinary ; In Situ Nick-End Labeling/veterinary ; Membrane Potential, Mitochondrial ; Microscopy, Confocal/veterinary ; Microscopy, Fluorescence/veterinary ; Mitochondria/*metabolism ; Nuclear Transfer Techniques/*veterinary ; Reactive Oxygen Species/*metabolism

We compared the efficiency of cloning goat using human lactoferrin (hLF) with genetically modified donor cells marked by single (Neo(r)) or double (Neo(r)/GFP) markers. Single marker expression vector (pBLC14) or dual markers expression vector (pAPLM) was delivered to goat fetal fibroblasts (GFF), and then the transgenic GFF was used as donor cells to produce transgenic goats. Respectively, 58.8% (20/34) and 86.7% (26/30) resistant cell lines confirmed the transgenic integration by PCR. Moreover, pAPLM cells lines were subcultured with several passages, only 20% (6/30) cell lines was observed fluorescence from each cell during the cell passage. Somatic cell nuclear transfer using the donor cells harbouring pBLC14 or pAPLM construct, resulting in a total of 806 reconstructed embryos, a pregnancy rate at 35 d (53.8%, 39.1%) and 60 d (26.9%, 21.7%), and an offspring birth rate (1.9%, 1.4%) with 5 and 7 newborn cloned goats, respectively. Transgene was confirmed by PCR and southern-blot in all cloned offspring. There were no significant differences at the reconstructed embryo fusion rates, pregnancy rates and the birth rate (P > 0.05) between single and double markers groups. The Neo(r)/GFP double markers could improve the reliability for accurately and efficiently selecting the genetically modified donor cells. No adverse effect was observed on the efficiency of transgenic goat production by SCNT using somatic cells transfected with double (Neo(r)/GFP) markers vector.
Animals ; Animals, Genetically Modified ; genetics ; Cloning, Molecular ; Cloning, Organism ; methods ; veterinary ; Fetus ; Fibroblasts ; cytology ; Genetic Markers ; Goats ; embryology ; genetics ; Green Fluorescent Proteins ; genetics ; Humans ; Lactoferrin ; biosynthesis ; genetics ; Neomycin ; Nuclear Transfer Techniques ; veterinary ; Recombinant Proteins ; biosynthesis ; genetics ; Transfection ; veterinary

Recently, we reported the three wolves cloning with normal karyotype from somatic cells of endangered male gray wolves (Canis lupus), but one wolf had female external genitalia. In this study, we conducted further clinical, histological, and genetic analyses. This cloned wolf had a normal uterus but developed ovotestis. Through molecular analysis of the SRY gene, a mutation in the coding sequence of SRY gene could be excluded as a cause of intersexuality. This is the first report of a cloned wolf with a 78, XY ovotesticular disorder affecting sexual development characterized by bilateral ovotestes.
Animals ; Cloning, Organism/*veterinary ; Female ; Karyotyping ; Mutation ; Nuclear Transfer Techniques/*veterinary ; Ovotesticular Disorders of Sex Development/pathology/*veterinary ; *Wolves