title: Production of cloned and transgenic embryos using buffalo (Bubalus bubalis) embryonic stem cell-like cells isolated from in vitro fertilized and cloned blastocysts.
abstract: Here, we report the isolation and characterization of embryonic stem (ES) cell-like cells from cloned blastocysts, generated using fibroblasts derived from an adult buffalo (BAF). These nuclear transfer embryonic stem cell-like cells (NT-ES) grew in well-defined and dome-shaped colonies. The expression pattern of pluripotency marker genes was similar in both NT-ES and in vitro fertilization (IVF) embryo-derived embryonic stem cell-like cells (F-ES). Upon spontaneous differentiation via embryoid body formation, cells of different morphology were observed, among which predominant were endodermal-like and epithelial-like cell types. The ES cell-like cells could be passaged only mechanically and did not form colonies when plated as single cell suspension at different concentrations. When F-ES cell-like, NT-ES cell-like, and BAF cells of same genotype were used for hand-made cloning (HMC), no significant difference (p > 0.05) was observed in cleavage and blastocyst rate. Following transfer of HMC embryos to synchronized recipients, pregnancies were established only with F-ES cell-like and BAF cell-derived embryos, and one live calf was born from F-ES cell-like cells. Further, when transfected NT-ES cell-like cells and BAF were used for HMC, no significant difference (p > 0.05) was observed between cleavage and blastocyst rate. In conclusion, here we report for the first time the derivation of ES cell-like cells from an adult buffalo, and its genetic modification. We also report the birth of a live cloned calf from buffalo ES cell-like cells.
authors.affiliation: Embryo Biotechnology Lab, Animal Biotechnology Centre, National Dairy Research Institute, Karnal, India.
authors.forename: Aman
authors.initials: A
authors.lastname: George
authors.forename: Ruchi
authors.initials: R
authors.lastname: Sharma
authors.forename: Karn P
authors.initials: KP
authors.lastname: Singh
authors.forename: Sudeepta K
authors.initials: SK
authors.lastname: Panda
authors.forename: Suresh K
authors.initials: SK
authors.lastname: Singla
authors.forename: Prabhat
authors.initials: P
authors.lastname: Palta
authors.forename: Radhaysham
authors.initials: R
authors.lastname: Manik
authors.forename: Manmohan S
authors.initials: MS
authors.lastname: Chauhan
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified / Q000196:embryology*; D001755:Blastocyst / Q000166:cytology*; D002020:Buffaloes / Q000196:embryology*; D002454:Cell Differentiation; D019976:Cloning, Organism / Q000379:methods*; D004624:Embryo Transfer; D004622:Embryo, Mammalian / Q000166:cytology*; D047108:Embryonic Development / Q000502:physiology; D053595:Embryonic Stem Cells / Q000166:cytology*; D005260:Female; D005307:Fertilization in Vitro / Q000379:methods*; D008297:Male; D053652:Nuclear Transfer Techniques; D011247:Pregnancy