title: Serial cloning of pigs by somatic cell nuclear transfer: restoration of phenotypic normality during serial cloning.
abstract: Somatic cell nuclear transfer (scNT) is a useful way to create cloned animals. However, scNT clones exhibit high levels of phenotypic instability. This instability may be due to epigenetic reprogramming and/or genomic damage in the donor cells. To test this, we produced transgenic pig fibroblasts harboring the truncated human thrombopoietin (hTPO) gene and used them as donor cells in scNT to produce first-generation (G1) cloned piglets. In this study, 2,818 scNT embryos were transferred to 11 recipients and five G1 piglets were obtained. Among them, a clone had a dimorphic facial appearance with severe hypertelorism and a broad prominent nasal bridge. The other clones looked normal. Second-generation (G2) scNT piglets were then produced using ear cells from a G1 piglet that had an abnormal nose phenotype. We reasoned that, if the phenotypic abnormality of the G1 clone was not present in the G2 and third-generation (G3) clones, or was absent in the G2 clones but reappeared in the G3 clones, the phenotypic instability of the G1 clone could be attributed to faulty epigenetic reprogramming rather than to inherent/accidental genomic damage to the donor cells. Blastocyst rates, cell numbers in blastocyst, pregnancy rates, term placenta weight and ponderal index, and birth weight between G1 and G2 clones did not differ, but were significantly (P < 0.05) lower than control age- and sex-matched piglets. Next, we analyzed global methylation changes during development of the preimplantation embryos reconstructed by donor cells used for the production of G1 and G2 clones and could not find any significant differences in the methylation patterns between G1 and G2 clones. Indeed, we failed to detect the phenotypic abnormality in the G2 and G3 clones. Thus, the phenotypic abnormality of the G1 clone is likely to be due to epigenetic dysregulation. Additional observations then suggested that expression of the hTPO gene in the transgenic clones did not appear to be the cause of the phenotypic abnormality in the G1 clones and that the abnormality was acquired by only a few of the G1 clone's cells during its gestational development.
authors.affiliation: Division of Applied Life Science, College of Agriculture and Life Science, Gyeongsang National University, Jinju, GyeongNam, South Korea.
authors.forename: Seong-Keun
authors.initials: SK
authors.lastname: Cho
authors.forename: Jae-Hwan
authors.initials: JH
authors.lastname: Kim
authors.forename: Jong-Yi
authors.initials: JY
authors.lastname: Park
authors.forename: Yun-Jung
authors.initials: YJ
authors.lastname: Choi
authors.forename: Jae-Il
authors.initials: JI
authors.lastname: Bang
authors.forename: Kyu-Chan
authors.initials: KC
authors.lastname: Hwang
authors.forename: Eun-Jeong
authors.initials: EJ
authors.lastname: Cho
authors.forename: Sea-Hwan
authors.initials: SH
authors.lastname: Sohn
authors.forename: Sang Jun
authors.initials: SJ
authors.lastname: Uhm
authors.forename: Deog-Bon
authors.initials: DB
authors.lastname: Koo
authors.forename: Kyung-Kwang
authors.initials: KK
authors.lastname: Lee
authors.forename: Teoan
authors.initials: T
authors.lastname: Kim
authors.forename: Jin-Hoi
authors.initials: JH
authors.lastname: Kim
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D019976:Cloning, Organism / Q000379:methods / Q000662:veterinary*; D004249:DNA Damage; D017931:DNA Primers / Q000235:genetics; D005260:Female; D042822:Genomic Instability; D006801:Humans; D008297:Male; D008565:Membrane Proteins / Q000235:genetics; D053652:Nuclear Transfer Techniques / Q000662:veterinary*; D010641:Phenotype; D010920:Placenta / Q000002:abnormalities; D011247:Pregnancy; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics; D034421:Sus scrofa / Q000235:genetics; D013926:Thrombopoietin / Q000235:genetics; D060170:Uroplakin II
chemical_list: D017931:DNA Primers; D008565:Membrane Proteins; D011994:Recombinant Proteins; C554658:UPK2 protein, human; D060170:Uroplakin II; D013926:Thrombopoietin