Somatic cell nuclear transfer (SCNT) is considered to be a critical tool for propagating valuable animals. To determine the productivity calves resulting from embryos derived with different culture media, enucleated oocytes matured in vitro were reconstructed with fetal fibroblasts, fused, and activated. The cloned embryos were cultured in modified synthetic oviduct fluid (mSOF) or a chemically defined medium (CDM) and developmental competence was monitored. After 7 days of culturing, the blastocysts were transferred into the uterine horn of estrus-synchronized recipients. SCNT embryos that were cultured in mSOF or CDM developed to the blastocysts stages at similar rates (26.6% vs. 22.5%, respectively). A total of 67 preimplantational stage embryos were transferred into 34 recipients and six cloned calves were born by caesarean section, or assisted or natural delivery. Survival of transferred blastocysts to live cloned calves in the mSOF and the CDM was 18.5% (to recipients), 9.6% (to blastocysts) and 42.9% (to recipients), 20.0% (to blastocysts), respectively. DNA analysis showed that all cloned calves were genetically identical to the donor cells. These results demonstrate that SCNT embryos cultured in CDM showed higher viability as judged by survival of the calves that came to term compared to blastocysts derived from mSOF cultures.
Animals ; Blastocyst/physiology ; *Cattle ; Cloning, Organism/methods/*veterinary ; Culture Media/chemistry ; Embryo Culture Techniques ; Embryo Transfer ; Embryonic Development ; Female ; Fertilization in Vitro/*veterinary ; Nuclear Transfer Techniques/*veterinary ; Pregnancy

In order to enhance the efficiency of sheep somatic cell nuclear transfer, we used a chemically assisted enucleation with colchicine to study the effects of the concentration of colchicine, the incubation time of oocytes in colchicine and the maturation time of oocytes on the enucleation rates and the development of reconstructed embryos. The results showed that 1) there were no significant differences in the rates of cytoplast protrusion and enucleation between oocytes that were incubated in colchicine (0.4 microg/mL) for 0.5 h and oocytes that were incubated in colchicine (0.4 microg/mL) for 1 h, and the rate of cytoplast protrusion can be 85.4% while the rate of cytoplast enucleation is 100%. 2) There was no significant difference in oocyte enucleation between oocytes treated with medium containing 0.2 microg/mL colchicine for 0.5 h and oocytes treated with medium containing 0.4 microg/mL colchicine for 0.5 h. 3) A maturation time of 18-23 h did not affect the rates of cytoplast protrusion and enucleation by chemically assisted enucleation, whereas the rate of enucleation of oocytes by blind enucleation was found to decrease with a prolonged incubation time. 4) The development rates of reconstructed embryos could not be influenced by these two enucleation methods, increased from oocytes matured for 21-23 h. These results demonstrate that sheep oocytes can be enucleated fast and effectively by optimized colcholine chemically assisted enucleation, which can enhance the enucleation rate of sheep oocytes and the early development of reconstructed embryos in vitro.
Animals ; Cloning, Organism ; methods ; Colchicine ; pharmacology ; Embryo, Mammalian ; embryology ; Female ; Nuclear Transfer Techniques ; veterinary ; Oocytes ; cytology ; drug effects ; Sheep

To optimize program of bovine somatic nuclear transfer, we used two different enucleation procedures (by Spindle-view system & Hoechst 33342 staining), two different procedures to introduce donor nuclei (by ooplasm microinjection & electrofusion), and three different group electrofusion parameters (group 1: 1.9 kV/cm, 10 micros, two; group 2: 1.5 kV/cm, 25 micros, two; group 3: 0.6 kV/cm, 100 micros, one) to reconstruct bovine cloned embryos. The cleavation rates and blastocyst development rates of cloned embryos were used to assess the efficiency of different operational procedure. Finally, the best combination of operational procedure, that the spindle-viewer system was used for oocytes enucleating, and donor cell was electrofused into ooplasm by electrical pulse (1.9 kV/cm, 10 micros, two) to reconstruct bovine cloned embryos. Then the excellent blastocysts were transferred to fosters for producing cloned cattle 80 high-quality cloned blastocysts were transferred into 33 fosters, two cloned calves were produced. According to the results, the optimized program could be used to produce cloned cattle.
Animals ; Cattle ; Cell Nucleus ; physiology ; Cloning, Organism ; veterinary ; Embryo Transfer ; methods ; Embryo, Mammalian ; cytology ; physiology ; Female ; Microinjections ; Nuclear Transfer Techniques ; veterinary ; Oocytes ; cytology ; physiology

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

The preimplantation development competences of somatic cell nuclear transfer (SCNT) embryos reconstructed with enuleated goat (Capra hircus) Metaphase II (MII) oocytes matured in vivo and whole cells derived from adult fibroblasts of several mammalian species (goat, boer goat, bovine, tahr, panda) and human patient were evaluated. Results obtained from our experiments revealed that these reconstructed SCNT embryos could complete preimplantation development to form blastocysts. The fusion rate and blastocyst rate of intra-species SCNT embryos (Capra hircus as control) was 78.67 (557/708); 56.29% (264/469), that of sub-species or inter-species SCNT embryos were: boer goat 78.18% (541/692); 33.90% (40/118), bovine 70.53% (146/207); 22.52% (25/111), tahr 53.51% (61/114); 5.26% (3/570), panda 79.82% (1159/1452); 8.35% (75/898) and human 68.76% (317/461); 5.41% (16/296), respectively. It is concluded that (1) there are no relationships between fusion rate and relativeness of the recipient cytoplasm to nucleus donor cells, (2) cytoplast of the goat MII oocyte can support the preimplantation development of SCNT embryos reconstructed with nucleus from other species, (3) the blastocyst rate of close relative inter-species SCNT embryos is higher than that of distant relative inter-species SCNT embryos.
Animals ; Cattle ; Cloning, Organism ; veterinary ; Embryo Culture Techniques ; methods ; veterinary ; Embryo, Mammalian ; physiology ; Embryonic Development ; physiology ; Female ; Fibroblasts ; cytology ; Goats ; embryology ; genetics ; Humans ; Nuclear Transfer Techniques ; veterinary ; Oocytes ; cytology ; physiology ; Pregnancy

Recently, the world's first transgenic dogs were produced by somatic cell nuclear transfer. However, cellular senescence is a major limiting factor for producing more advanced transgenic dogs. To overcome this obstacle, we rejuvenated transgenic cells using a re-cloning technique. Fibroblasts from post-mortem red fluorescent protein (RFP) dog were reconstructed with in vivo matured oocytes and transferred into 10 surrogate dogs. One puppy was produced and confirmed as a re-cloned dog. Although the puppy was lost during birth, we successfully established a rejuvenated fibroblast cell line from this animal. The cell line was found to stably express RFP and is ready for additional genetic modification.
Animals ; Animals, Genetically Modified ; Cloning, Organism/methods/*veterinary ; Dogs/*genetics ; Female ; Gastrointestinal Tract/metabolism ; Gene Expression Regulation ; Kidney/metabolism ; Liver/metabolism ; Luminescent Proteins/*genetics/metabolism ; Lung/metabolism ; Male ; Myocardium/metabolism ; Nuclear Transfer Techniques/veterinary ; Spleen/metabolism ; Trachea/metabolism

UNLABELLEDThis study is conducted to explore an effective culture method for supporting the embryo development. The cattle fetal ear fibroblasts and the goat fetal ear fibroblasts are transplanted into the enucleated cattle oocytes separately by oocyte intraplasmic nuclear injection method to construct bovine cloned embryos and goat-bovine cloned embryos. The embryos are first cultivated in modified charles rosenkrans 2 amino acid medium (mCR2aa) and modified synthetic oviduct fluid medium (mSOF) separately. Then BSA (8 mg/mL) or FBS (10%) can be added to mSOF according to the different culture period. The supplements and orders, added during the first three days and after three days are as follow: BSA and BSA, BSA and FBS, FBS and BSA, FBS and FBS. On the basis of the cleavage rate, 8/16-cell rate, blastocysts rate and total cell number of blastocysts, the best culture way can be screened out.

RESULTFirst, cleavage rate, 8/16-cell rate, blastocysts rate and total cell number of blastocysts, cultivated in mSOF solution are all higher than those cultivated in mCR2aa( P < 0.05). Second, the cleavage rate and 8/16-cell rate, adding BSA and FBS into mSOF, are in turn 79.8% +/- 7.1%, 49.7% +/- 3.5%, 21.5% +/- 1.8%, and 115.2 +/- 4.3 in bovine cloned embryo, and 40.1% +/- 6.3%, 29.2% +/- 2.0%, 13.4% +/- 2.1% and 100.1 +/- 3.0 in goat-bovine cloned embryo, which are significant higher than other culture groups (P < 0.05).

CONCLUSIONThe goat-bovine cloned embryo can be cultivated by the optimized culture measure of bovine cloned embryo. The best culture ways of bovine cloned embryo and goat-bovine cloned embryo are all to use mSOF supplemented BSA in the first three days and then use mSOF supplemented FBS in the next five days.

Animals ; Cattle ; embryology ; physiology ; Cells, Cultured ; Cloning, Organism ; veterinary ; Ear, External ; cytology ; Embryo Culture Techniques ; methods ; veterinary ; Embryonic Development ; Fibroblasts ; cytology ; transplantation ; Goats ; embryology ; physiology ; Nuclear Transfer Techniques ; Oocytes ; cytology

In producing transgenic livestock, selectable marker genes (SMGs) are usually used to screen transgenic cells from numerous normal cells. That results in SMGs integrating into the genome and transmitting to offspring. In fact, SMGs could dramatically affect gene regulation at integration sites and also make the safety evaluation of transgenic animals complicated. In order to determine the deletion time and methods in the process of producing transgenic goats, the feasibility of deleting SMGs was explored by Cre/LoxP before or after somatic cell cloning. In addition, we compared the efficiency of protein transduction with plasmids co-transduction. We could delete 43.9% SMGs after screening out the transgenic cell clones, but these cells could not be applied to somatic cells cloning because of serious aging after two gene modifications. The SMG-free cells suitable for nuclear transfer were accessible by using the cells of transgenic goats, but this approach was more time consuming. Finally, we found that the Cre plasmid could delete SMGs with an efficiency of 7.81%, but about 30% in SMG-free cells had sequences of Cre plasmid. Compared with Cre plasmid, the integration of new exogenous gene could be avoided by TAT-CRE protein transduction, and the deletion rate of TAT-CRE transduction was between 43.9 and 72.8%. Therefore, TAT-Cre transduction could be an effective method for deleting selectable marker genes.
Animals ; Animals, Genetically Modified ; genetics ; Cloning, Organism ; veterinary ; Gene Knockout Techniques ; Gene Targeting ; methods ; Genes, Reporter ; Genetic Engineering ; Genetic Vectors ; genetics ; Goats ; genetics ; Integrases ; chemistry ; metabolism ; Recombination, Genetic ; Transgenes ; genetics

In order to research developmental competence of transgenic somatic cell by serial nuclear transplantation, goat cloned embryos were compared with recloned embryos in ability of in vitro development. Fetal fibroblasts including human finger-domain lacking t-PA gene was microinjected into cytoplasm of the MII oocytes. Goat embryos (G0) were cloned by this procedure. A single blastomere from 16 - 64-cell goat cloned embryos (G0) was microinjected into Intracytoplasm of the MII oocytes. Goat embryos (G) were cloned by this procedure. Goat embryos (G2, G3) were recloned by using 16 - 64-cell recloned embryos. The developmental time of donor embryo affected the developmental rate of recloned embryos (G1, G2). The results show: the cleavage rate of cloned embryos (G0) (76.45% +/- 1.17%) was no difference significantly with recloned embryos (G1 G2 G3) (72.18% +/- 1.97%, 76.05% +/- 2.38%, 75.99% +/- 2.84%); the developmental rate of morulae and blastocysts of cloned embryos (47.20% +/- 2.93%, 11.00% +/- 1.42%) were higher than these of recloned embryos(34.99% +/- 2.66%, 28.23% +/- 2.00%, 23.34% +/- 1.99%) (3.87% +/- 0.67%, 2.08% +/- 1.66%, 0); the morulae rate(29.57% +/- 1.53%, 24.43% +/- 1.87%) and blastocysts rate(1.96% +/- 1.31%, 2.01% +/- 1.34%) of recloned embryos (G1 G2) from 16-cell recloned embryos were lower than those(34.32% +/- 1.31%, 29.76% +/- 1.66% and 3.86% +/- 1.03%, 3.48% +/- 0.34% )from 32 - 64-cell recloned embryos (P > 0.05). In conclusion, nuclear transfer embryos should not were recloned mostly; and the embryos recloned by using 32 - 64-cell embryos achieved higher developmental ability compared with using 16-cell embryos.
Animals ; Animals, Genetically Modified ; Blastomeres ; cytology ; physiology ; Cloning, Organism ; methods ; veterinary ; Embryo Culture Techniques ; Embryo, Mammalian ; cytology ; Female ; Fibroblasts ; cytology ; Gene Deletion ; Goats ; Humans ; Nuclear Transfer Techniques ; veterinary ; Oocytes ; cytology ; physiology ; Pregnancy ; RING Finger Domains ; genetics ; physiology ; Tissue Plasminogen Activator ; genetics ; metabolism