Nna miniature inbred pigs have been bred since the 1980s from full and half siblings. As unique, highly miniature inbred pigs, Banna miniature inbred pigs can serve as large mammalian models with high homozygotic genes and clear genetic background [1,2]. Given their similar anatomical and physiological features to humans, these animals can be used in various biomedical studies, including disease models, 842-07-9 transgenesis, genomics, and xenotransplantation for medical research [3]. Some special traits also appear in inbreeding, such as blindness, deafness, spinal column bend, maxilla defect, and tumor. This particular phenotype provides valuable resources for studying relative human diseases. However, these individuals are hardly reproducible because of their impaired fertility or lethality. Thus, establishing a cloning system is essential to reproduce Banna miniature inbred pigs with unique traits for application to studies in various fields. Somatic cell nuclear transfer (SCNT) is an important method of breeding quality varieties, expanding groups, and preserving endangered species [4]. This method was successfully applied incalf [5], mouse [6], goat [7], pig [8], rabbit [9], cat [10], rat [11], horse [12], mule [13], dog [14], ferret [15], buffalo [16], and camel [17] since the world’s first cloned sheep was obtained in 1996 [18]. Feasible SCNT procedures were established in pig. However, miniature pigs, such as the JI 101 National Institutes of Health miniature pigs [19] and Clawn miniature pigs, have low cloning efficiency [20]. Under such circumstances, several studies focused on nuclear donor cells, which are generally believed to affect the cloning efficiency in mammals. In cattle, fetal fibroblasts are reportedly more effective than newborn fibroblasts [21]. In pig, fetal fibroblasts are more effective than adult fibroblasts as well as cumulus and oviduct cells [22]. Cell cycle synchronization through differentiation induction enables the effective production of cloned pigs [23]. In mouse, the appropriate combinations of cell type and genotype may improve the efficiency of somatic 
cell cloning and fetal survival after embryo transfer [24]. However, the cloning process and 
efficiency in Banna miniature inbred pigs remain unclear. The present study aims to establish the nuclear transfer technology system of 10457188 Banna miniature inbred pig and to investigateCloning of Banna Miniature Inbred Pigthe effect of different donor cells, i.e., fetal, newborn, and adult fibroblasts, on the developmental competence of SCNT embryos as well as on the cloning efficiency of this pig.Materials and MethodsAll animal experiments were performed with the approval of the Animal Care Committee of Yunnan Agricultural University, China.ChemicalsUnless otherwise stated, all chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA).Preparation of Donor CellsFetuses (47 days old) isolated from the 22nd generation in the No. 133-family of Banna miniature inbred pig were washed three times with phosphate-buffered saline. After removing the head, limbs, and viscera, the fetuses were minced and digested in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) containing 20 fetal bovine serum (FBS; Hyclone), 1 penicillin-streptomycin, and 1 mg/mL Collagenase IV for 4 h at 37uC. The cells were centrifuged at 1000 rpm for 5 min, suspended in DMEM supplemented 26001275 with 10 FBS and 1 penicillin-streptomycin, and then cultured in a flask until grown to 90 confluence. The cells.Nna miniature inbred pigs have been bred since the 1980s from full and half siblings. As unique, highly miniature inbred pigs, Banna miniature inbred pigs can serve as large mammalian models with high homozygotic genes and clear genetic background [1,2]. Given their similar anatomical and physiological features to humans, these animals can be used in various biomedical studies, including disease models, transgenesis, genomics, and xenotransplantation for medical research [3]. Some special traits also appear in inbreeding, such as blindness, deafness, spinal column bend, maxilla defect, and tumor. This particular phenotype provides valuable resources for studying relative human diseases. However, these individuals are hardly reproducible because of their impaired fertility or lethality. Thus, establishing a cloning system is essential to reproduce Banna miniature inbred pigs with unique traits for application to studies in various fields. Somatic cell nuclear transfer (SCNT) is an important method of breeding quality varieties, expanding groups, and preserving endangered species [4]. This method was successfully applied incalf [5], mouse [6], goat [7], pig [8], rabbit [9], cat [10], rat [11], horse [12], mule [13], dog [14], ferret [15], buffalo [16], and camel [17] since the world’s first cloned sheep was obtained in 1996 [18]. Feasible SCNT procedures were established in pig. However, miniature pigs, such as the National Institutes of Health miniature pigs [19] and Clawn miniature pigs, have low cloning efficiency [20]. Under such circumstances, several studies focused on nuclear donor cells, which are generally believed to affect the cloning efficiency in mammals. In cattle, fetal fibroblasts are reportedly more effective than newborn fibroblasts [21]. In pig, fetal fibroblasts are more effective than adult fibroblasts as well as cumulus and oviduct cells [22]. Cell cycle synchronization through differentiation induction enables the effective production of cloned pigs [23]. In mouse, the appropriate combinations of cell type and genotype may improve the efficiency of somatic cell cloning and fetal survival after embryo transfer [24]. However, the cloning process and efficiency in Banna miniature inbred pigs remain unclear. The present study aims to establish the nuclear transfer technology system of 10457188 Banna miniature inbred pig and to investigateCloning of Banna Miniature Inbred Pigthe effect of different donor cells, i.e., fetal, newborn, and adult fibroblasts, on the developmental competence of SCNT embryos as well as on the cloning efficiency of this pig.Materials and MethodsAll animal experiments were performed with the approval of the Animal Care Committee of Yunnan Agricultural University, China.ChemicalsUnless otherwise stated, all chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA).Preparation of Donor CellsFetuses (47 days old) isolated from the 22nd generation in the No. 133-family of Banna miniature inbred pig were washed three times with phosphate-buffered saline. After removing the head, limbs, and viscera, the fetuses were minced and digested in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) containing 20 fetal bovine serum (FBS; Hyclone), 1 penicillin-streptomycin, and 1 mg/mL Collagenase IV for 4 h at 37uC. The cells were centrifuged at 1000 rpm for 5 min, suspended in DMEM supplemented 26001275 with 10 FBS and 1 penicillin-streptomycin, and then cultured in a flask until grown to 90 confluence. The cells.
