Ncreasing age. Several studies reported SCNT attempts in miniature pig. The donor cells of these pigs are all derived only from fetal fibroblasts. The cloning efficiency of the Chinese Bama miniature pig is 0.11 [1/870 (no. offspring/no. embryos transferred in the recipients; 68181-17-9 biological activity similarly hereinafter)] [41]. The efficiencies of producing cloned Potbelly miniature pigs from the lung and kidney of male newborn Meishan pigs as recipients are 8.57 and 3.57 (3/35 and 2/56), respectively [42]. Both the reconstructed embryos of Bama and Potbelly miniature inbred pigs were transferred at the two- to fourcell stage. Clawn miniature pig developed from cloned embryos was transferred to recipients after culturing for 6 h to 40 h and showed a cloning efficiency of 2.35 (2/85) [20]. Yucatan miniature pig was successfully cloned at an efficiency of 1.1 (7/ 631). Embryos were cultured for less than 1 h before being surgically transferred into the recipient [43]. The production efficiencies of cloned Nippon Institute for Biological Sciences strain miniature pigs using male and female fetal fibroblasts as nucleus donors range from 0.64 (2/314) to 0.9 (3/331) by transferring reconstructed embryos cultured for 1 day to 2 days into miniature and common domestic pigs [44]. In the National Institute of Health miniature inbred pig, the cloning efficiency isTable 5. Microsatellite analysis of cloned piglets derived from fetal fibroblasts.MarkerPCR Dye annealing name tempGenotypes of Recipient 1 2 93/101 285/287 142/152 193 108/110 101/103 99/109 168 117/123 139/151 95/109 3 91/Ornipressin site 97Cell line genotypes Genotypes of litter (BN133) 1 97 271/273 97 2 97 3 97 4 97 5 97 6 97 7 97 8S0026 S0070 S0155 S0226 SW122 SW24 SW72 SW830 SW840 SW857 SWFAM FAM FAM FAM FAM FAM FAM FAM FAM FAM FAM55 55 55 55 55 55 55 50 55 5597 271/273 142/152 183/195 108 103 97 180/184 135 139/155271/273 271/273 142 195 108 103 97 142 195 108 103271/273 271/273 271/273 142 195 108 103 97 142 195 108 103 97 142 195 108 103271/273 271/273 271/273 142 195 108 103 97 142 195 108 103 97 142 195 108 103152/156 142 183106/110 108 115 97 180 103 97 180/180/184 180/184 135 139 97 135 139180/184 180/184 180/184 135 139 97 135 139 97 135 139180/184 180/184 180/184 135 139 97 135 139 97 135 139125/129 135 143/153 139 93/119*For each microsatellite marker, genotype was determined by size (base pairs). Two numbers for each sample at each locus represent the PCR product size at that particular locus. *Litters 1,2,3 came from recipient 1; litters 4,5,6 came from recipient 2; and the rest came from recipient 3. doi:10.1371/journal.pone.0057728.tCloning of Banna Miniature Inbred PigTable 6. Microsatellite analysis of cloned piglets derived from newborn fibroblasts.MarkerDye namePCR annealing tempGenotypes of RecipientCell line genotypesGenotypes of litter 1 2 
93 273 142 195 108 115 98/110 185 125 154 97 3 93 273 142 195 108 115 98/110 185 125 154S0026 S0070 S0155 S0226 SW122 SW24 SW72 SW830 SW840 SW857 SWFAM FAM FAM FAM FAM FAM FAM FAM FAM FAM FAM55 55 
55 55 55 55 55 50 55 5597 273/283 152/158 181 112 103 106/108 177 125 14493 273 142 195 108 115 98/110 185 125 15493 273 142 195 108 115 98/110 185 125 154doi:10.1371/journal.pone.0057728.t1.3 (21/1610) using fetal fibroblasts as 1326631 donor cells and transferring on the same day of SCNT [19]. In several inbred mice that have not been cloned (such as C57BL/6 and C3H/He), the cloning efficiencies of cloned inbred strains are extremely low, similar to the DBA/2 and 129/Sv.Ncreasing age. Several studies reported SCNT attempts in miniature pig. The donor cells of these pigs are all derived only from fetal fibroblasts. The cloning efficiency of the Chinese Bama miniature pig is 0.11 [1/870 (no. offspring/no. embryos transferred in the recipients; similarly hereinafter)] [41]. The efficiencies of producing cloned Potbelly miniature pigs from the lung and kidney of male newborn Meishan pigs as recipients are 8.57 and 3.57 (3/35 and 2/56), respectively [42]. Both the reconstructed embryos of Bama and Potbelly miniature inbred pigs were transferred at the two- to fourcell stage. Clawn miniature pig developed from cloned embryos was transferred to recipients after culturing for 6 h to 40 h and showed a cloning efficiency of 2.35 (2/85) [20]. Yucatan miniature pig was successfully cloned at an efficiency of 1.1 (7/ 631). Embryos were cultured for less than 1 h before being surgically transferred into the recipient [43]. The production efficiencies of cloned Nippon Institute for Biological Sciences strain miniature pigs using male and female fetal fibroblasts as nucleus donors range from 0.64 (2/314) to 0.9 (3/331) by transferring reconstructed embryos cultured for 1 day to 2 days into miniature and common domestic pigs [44]. In the National Institute of Health miniature inbred pig, the cloning efficiency isTable 5. Microsatellite analysis of cloned piglets derived from fetal fibroblasts.MarkerPCR Dye annealing name tempGenotypes of Recipient 1 2 93/101 285/287 142/152 193 108/110 101/103 99/109 168 117/123 139/151 95/109 3 91/97Cell line genotypes Genotypes of litter (BN133) 1 97 271/273 97 2 97 3 97 4 97 5 97 6 97 7 97 8S0026 S0070 S0155 S0226 SW122 SW24 SW72 SW830 SW840 SW857 SWFAM FAM FAM FAM FAM FAM FAM FAM FAM FAM FAM55 55 55 55 55 55 55 50 55 5597 271/273 142/152 183/195 108 103 97 180/184 135 139/155271/273 271/273 142 195 108 103 97 142 195 108 103271/273 271/273 271/273 142 195 108 103 97 142 195 108 103 97 142 195 108 103271/273 271/273 271/273 142 195 108 103 97 142 195 108 103 97 142 195 108 103152/156 142 183106/110 108 115 97 180 103 97 180/180/184 180/184 135 139 97 135 139180/184 180/184 180/184 135 139 97 135 139 97 135 139180/184 180/184 180/184 135 139 97 135 139 97 135 139125/129 135 143/153 139 93/119*For each microsatellite marker, genotype was determined by size (base pairs). Two numbers for each sample at each locus represent the PCR product size at that particular locus. *Litters 1,2,3 came from recipient 1; litters 4,5,6 came from recipient 2; and the rest came from recipient 3. doi:10.1371/journal.pone.0057728.tCloning of Banna Miniature Inbred PigTable 6. Microsatellite analysis of cloned piglets derived from newborn fibroblasts.MarkerDye namePCR annealing tempGenotypes of RecipientCell line genotypesGenotypes of litter 1 2 93 273 142 195 108 115 98/110 185 125 154 97 3 93 273 142 195 108 115 98/110 185 125 154S0026 S0070 S0155 S0226 SW122 SW24 SW72 SW830 SW840 SW857 SWFAM FAM FAM FAM FAM FAM FAM FAM FAM FAM FAM55 55 55 55 55 55 55 50 55 5597 273/283 152/158 181 112 103 106/108 177 125 14493 273 142 195 108 115 98/110 185 125 15493 273 142 195 108 115 98/110 185 125 154doi:10.1371/journal.pone.0057728.t1.3 (21/1610) using fetal fibroblasts as 1326631 donor cells and transferring on the same day of SCNT [19]. In several inbred mice that have not been cloned (such as C57BL/6 and C3H/He), the cloning efficiencies of cloned inbred strains are extremely low, similar to the DBA/2 and 129/Sv.
