Strategies for the Derivation of Pluripotent Cells from Farm Animals

Abstract

Recent experiments demonstrated that forced expression of few critical genes drives conversion of a somatic into a pluripotent cell state. These induced pluripotent cells (iPS) were first generated from murine fibroblasts by Shinya Yamanaka's laboratory in 2006. By using retroviral vectors to express combinations of stemness genes, they identified Oct4, Sox2, Krueppel-like factor 4 and c-Myc as essential factors for reprogramming of somatic cells. Subsequent experiments applied this technology to human and rat fibroblasts, as well as other cell types and several groups showed that iPS can be generated by an even smaller number of transcription factors. The efficiency of conversion and maintenance of a pluripotent state can be supported by small molecules, such as valproic acid and specific pharmacological inhibitors. This technology is a milestone for a basic understanding of cell potency, cell fate and pathogenesis, as well as for development of cell therapies and potential applications in animal breeding.

Derivation and Characterization ofSleeping BeautyTransposon-Mediated Porcine Induced Pluripotent Stem Cells

“…After breeding of chimeric pigs, 2 out of 43 F1 offspring carried some of the reprogramming factors in their genome, suggesting germ line transmission [29]. However, current data indicate that reprogramming and in vitro culture conditions are deficient for induction and maintenance of pluripotency in porcine iPS cells [24,[30][31][32]. In contrast to other species, in the pig, the continuous expression of the exogenous transgenic transcription factors is still necessary to maintain the pluripotent phenotype of presumptive porcine iPS cells [25][26][27][28].…”

Genotype-Independent Transmission of Transgenic Fluorophore Protein by Boar Spermatozoa

“…1A) and three cloned transgenic boars (F0), carrying an Oct4-EGFP construct [39], [40], were analysed (Table 1). The transcriptional activity of the Oct4 promoter is restricted to the germline, and EGFP fluorescence was exclusively detected in blastomeres of cleavage-stage embryos, germline cells of genital ridge and testis [39], however, ejaculated spermatozoa have not been analysed before.…”

“…Two transposon transgenic boars [17] , [18] , each carrying three monomeric integrants of an ubiquitously active CMV early enhancer, chicken beta actin (CAGGS) promoter driven Venus construct ( Fig. 1A ) and three cloned transgenic boars (F0), carrying an Oct4-EGFP construct [39] , [40] , were analysed ( Table 1 ). The transcriptional activity of the Oct4 promoter is restricted to the germline, and EGFP fluorescence was exclusively detected in blastomeres of cleavage-stage embryos, germline cells of genital ridge and testis [39] , however, ejaculated spermatozoa have not been analysed before.…”

Trichostatin A-Mediated Epigenetic Transformation of Adult Bone Marrow-Derived Mesenchymal Stem Cells Biases theIn VitroDevelopmental Capability, Quality, and Pluripotency Extent of Porcine Cloned Embryos

“…Improved reprogrammability of transcriptional activity for nuclear genome of epigenetically modulated MSCs in the cells of preimplanted NT embryos turned out to be positively correlated with enhanced molecular quality of porcine cloned blastocysts assessed on the basis of their pluripotency extent, which was measured with the expression profiles identified for Oct4 and Nanog genes. A 38-kDa protein Oct4 (i.e., octamer-binding transcription factor 4) that is a member of the family of POU- (Pit-Oct-Unc-) domain and homeodomain transcription factors acts as a vital regulator of pluripotency extent, playing an important role in not only controlling preimplantation embryonic development, but also maintenance of ICM cell fate in blastocysts and pluripotency status of embryonic stem cells (ESCs) [ 68 – 70 ]. A 35-kDa protein designated as Nanog from Celtic/Irish mythical Tír na nÓg (Tir Na Nog; The Land of the Ever-Young) is another homeobox-containing transcription factor that represents the group of pivotal proteins modulating pluripotency degree [ 70 , 71 ].…”

“…A 38-kDa protein Oct4 (i.e., octamer-binding transcription factor 4) that is a member of the family of POU- (Pit-Oct-Unc-) domain and homeodomain transcription factors acts as a vital regulator of pluripotency extent, playing an important role in not only controlling preimplantation embryonic development, but also maintenance of ICM cell fate in blastocysts and pluripotency status of embryonic stem cells (ESCs) [ 68 – 70 ]. A 35-kDa protein designated as Nanog from Celtic/Irish mythical Tír na nÓg (Tir Na Nog; The Land of the Ever-Young) is another homeobox-containing transcription factor that represents the group of pivotal proteins modulating pluripotency degree [ 70 , 71 ]. The homeoprotein Nanog can act synergistically with Oct4 protein in retaining the pluripotent status of blastocyst-descended ICM and epiblast cells as well as in sustaining the undifferentiated status and ability for self-renewal of ESCs [ 71 , 72 ].…”