Cloning, Organism ; legislation & jurisprudence ; methods ; Ethics, Medical ; Genetic Techniques ; Humans ; Religion and Medicine ; Stem Cell Transplantation

In 2007, seven detector dogs were produced by somatic cell nuclear transfer using one nuclear donor dog, then trained and certified as excellent detector dogs, similar to their donor. In 2011, we crossed a cloned male and normal female by natural breeding and produced ten offspring. In this study, we investigated the puppies' temperaments, which we later compared with those of the cloned parent male. The results show that the cloned male had normal reproductive abilities and produced healthy offspring. All puppies completed narcotic detector dog training with a success rate for selection of 60%. Although the litter of cloned males was small in this study, a cloned male dog bred by natural mating produced puppies that later successfully completed the training course for drug detection. In conclusion, cloning an elite dog with superior genetic factors and breeding of the cloned dog was found to be a useful method to efficiently procure detector dogs.
Animals ; Breeding ; Clone Cells* ; Cloning, Organism ; Dogs* ; Female ; Humans ; Male* ; Methods ; Parents ; Temperament ; Tissue Donors

For the first time ever, the scientists of J. Craig Venter team have created actual self-replicating synthetic life. The research was just published in the Journal of Science on May 20, 2010. Although this news immediately brings the worry about the possible potential threat to biosecurity and biosafety as well as the ethical disputes, it yet indicates that mankind have made a new step forward in synthetic biology. In the time of post-genome era, we believe the advancement of synthetic biology that might affect or change the future life of human being will be widely used in energy, environment, materials, medication and many other fields.
Biological Science Disciplines ; methods ; Cloning, Organism ; Genome, Human ; genetics ; Human Genome Project ; Humans ; Synthetic Biology ; trends

T-vectors are widely used for cloning the polymerase chain reaction (PCR) products. However, the low conversion efficiency of a plasmid into the linear T-vector usually results in non-recombinants. Here, we designed a new plasmid pNBQ-T to easily select the recombinant colonies harboring PCR products. pNBQ-T plasmid, which contains a DsRed indicator gene between two Nt.BspQI restriction cassettes, each of which contains palindromic sequences susceptible to Nt.BspQI nickase (5′-GCTCTTCT^GAAGAGC-3′) at each end. Thus, this plasmid can be easily converted into T-vectors by a nickase (quadruple nicking), which results in two double strand breaks with 3′-thymidine overhangs. DsRed indicator gene, which is inserted between the restriction sites, helps identifying the PCR recombinants. Using this pNBQ-T plasmid the insertion efficiency of a PCR product was examined. We successfully identified white colony of the recombinants with the inserted myostatin promoter gene: the cloning efficiency was 93%. Therefore, this simple method utilizing pNBQ-T plasmid will serve as a useful and efficient technique for preparation of home-made T-vectors.
Clone Cells ; Cloning, Organism ; Deoxyribonuclease I* ; Methods ; Myostatin ; Plasmids ; Polymerase Chain Reaction

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

Xenotransplantation of porcine organs has the potential to overcome the acute shortage of human tissues and organs for human transplantation. Swine represents an ideal source of such organs owing to their anatomical and physiological similarities to human besides their plentiful supply. However, this procedure is also associated with a number of safety issues related to zoonotic infections. Among such zoonotically important pathogens, porcine endogenous viruses (PERVs) represent the most concerned virus as they persist asymptomatically and show germline transmission in pigs. They belong to gamma retroviruses and are of three types viruses: A, B and C. In the present study, PCR based cloning was performed with chromosomal DNA extracted from miniature pigs to analyze the envelope gene of PERVs. Amplified PCR fragments of about 1.5 Kb, covering the partial env gene, were cloned into pCR2.1-TOPO vectors and sequenced. A total of 51 env clones were obtained from two miniature pigs, types M149 and T1111. Phylogenetic analysis of these genes revealed the presence of only PERV type A and B in the proportion of 45% and 55%, respectively. Among these, 9 clones had the correct open reading frame: eight were PERV type A and one PERV type B. Since both these PERV types are polytropic and have the capacity to infect human cells, our data raise a concern that proviral PERVs might have the potential to generate infectious viruses during or after xenotransplantation in humans.
Clone Cells ; Cloning, Molecular* ; Cloning, Organism ; DNA ; Genes, env ; Humans ; Methods ; Open Reading Frames ; Polymerase Chain Reaction ; Retroviridae ; Swine* ; Transplantation, Heterologous ; Zoonoses

Xenotransplantation of porcine organs has the potential to overcome the acute shortage of human tissues and organs for human transplantation. Swine represents an ideal source of such organs owing to their anatomical and physiological similarities to human besides their plentiful supply. However, this procedure is also associated with a number of safety issues related to zoonotic infections. Among such zoonotically important pathogens, porcine endogenous viruses (PERVs) represent the most concerned virus as they persist asymptomatically and show germline transmission in pigs. They belong to gamma retroviruses and are of three types viruses: A, B and C. In the present study, PCR based cloning was performed with chromosomal DNA extracted from miniature pigs to analyze the envelope gene of PERVs. Amplified PCR fragments of about 1.5 Kb, covering the partial env gene, were cloned into pCR2.1-TOPO vectors and sequenced. A total of 51 env clones were obtained from two miniature pigs, types M149 and T1111. Phylogenetic analysis of these genes revealed the presence of only PERV type A and B in the proportion of 45% and 55%, respectively. Among these, 9 clones had the correct open reading frame: eight were PERV type A and one PERV type B. Since both these PERV types are polytropic and have the capacity to infect human cells, our data raise a concern that proviral PERVs might have the potential to generate infectious viruses during or after xenotransplantation in humans.
Clone Cells ; Cloning, Molecular* ; Cloning, Organism ; DNA ; Genes, env ; Humans ; Methods ; Open Reading Frames ; Polymerase Chain Reaction ; Retroviridae ; Swine* ; Transplantation, Heterologous ; Zoonoses

The remarkable potential of embryonic stem (ES) cells is their ability to develop into many different cell types. ES cells make it possible to treat patients by transplanting specialized healthy cells derived from them to repair damaged and diseased cells or tissues, known as "stem cell therapy". However, the issue of immunocompatibility is one of considerable significance in ES cell transplantation. One approach to overcome transplant rejection of human ES (hES) cells is to derive hES cells from nuclear transfer of the patient's own cells. This concept is known as "therapeutic cloning". In this review, we describe the derivations of ES cells and cloned ES cells by somatic cell nuclear transfer, and their potential applications in transplantation medicine.
Animals ; Cell Culture Techniques/*methods ; Cloning, Organism/*methods ; Embryo/cytology ; Embryo Culture Techniques ; Humans ; Pluripotent Stem Cells/*cytology/immunology ; Stem Cell Transplantation/*methods

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

In this study, we evaluated the development potential of caprine mammary gland epithelial cells (CMGECs) after transfection and nuclear transfer into enucleated, ovulated oocytes. We first isolated CMGECs from udders of lactating goats which were transfected with expression plasmid for human lacterrin and selected by G418. Then we chose sixteen neomycin resistant lines and induced them with prolactin for the expression of human lactoferrin checked by Western blotting. The donor cells, expressing human lactoferrin of 75 kD, were fused and activated with enucleated ovulated oocytes. Pronuclear-stage reconstructed embryos were transferred into the oviducts of 16 recipient goats. There were fourteen (87.5%), thirteen (81.3%), and ten (62.5%) pregnancies confirmed pregnant by ultrasound on Day 30, 60, and 90, respectively. Three recipients carried the pregnancies to term and delivered one goat each. Nested PCR-RFLP analysis confirmed that all of the kids were clones of the donor cells. These results demonstrated that CMGECs after transfection remain totipotent for nuclear transfer.
Animals ; Cloning, Organism ; methods ; Epithelial Cells ; cytology ; Female ; Goats ; Humans ; Lactoferrin ; biosynthesis ; Mammary Glands, Animal ; cytology ; Nuclear Transfer Techniques ; Pregnancy ; Transfection