RESUMEN

Pig induced pluripotent stem cells (piPSCs) offer a great opportunity and a number of advantages in the generation of transgenic animals. These immortalized cells can undergo multiple rounds of genetic modifications (e.g., gene knock-in, knockout) and selection leading to animals that have optimized traits of biomedical or agricultural interests. In this chapter we describe the production and characterization of piPSCs, microinjection of piPSCs into embryos, embryo transfer and production of chimeric animals based on successful protocols.

Asunto(s)

Reprogramación Celular , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Animales , Técnicas de Cultivo de Célula , Transferencia de Embrión , Embrión de Mamíferos/citología , Femenino , Vectores Genéticos/genética , Inmunohistoquímica , Microinyecciones , Embarazo , Porcinos , Factores de Transcripción/genética , Transducción Genética

RESUMEN

Ethical and moral issues rule out the use of human induced pluripotent stem cells (iPSCs) in chimera studies that would determine the full extent of their reprogrammed state, instead relying on less rigorous assays such as teratoma formation and differentiated cell types. To date, only mouse iPSC lines are known to be truly pluripotent. However, initial mouse iPSC lines failed to form chimeric offspring, but did generate teratomas and differentiated embryoid bodies, and thus these specific iPSC lines were not completely reprogrammed or truly pluripotent. Therefore, there is a need to address whether the reprogramming factors and process used eventually to generate chimeric mice are universal and sufficient to generate reprogrammed iPSC that contribute to chimeric offspring in additional species. Here we show that porcine mesenchymal stem cells transduced with 6 human reprogramming factors (POU5F1, SOX2, NANOG, KLF4, LIN28, and C-MYC) injected into preimplantation-stage embryos contributed to multiple tissue types spanning all 3 germ layers in 8 of 10 fetuses. The chimerism rate was high, 85.3% or 29 of 34 live offspring were chimeras based on skin and tail biopsies harvested from 2- to 5-day-old pigs. The creation of pluripotent porcine iPSCs capable of generating chimeric offspring introduces numerous opportunities to study the facets significantly affecting cell therapies, genetic engineering, and other aspects of stem cell and developmental biology.

Asunto(s)

Quimera/embriología , Células Madre Pluripotentes Inducidas/citología , Sus scrofa , Estructuras Animales/citología , Estructuras Animales/metabolismo , Animales , Animales Recién Nacidos/anomalías , Animales Recién Nacidos/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Blastocisto/citología , Diferenciación Celular/genética , Quimera/anomalías , Quimera/metabolismo , Cuerpos Embrioides/citología , Proteínas Fetales/genética , Feto/citología , Feto/metabolismo , Expresión Génica/genética , Proteínas de Homeodominio/genética , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/trasplante , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Células Madre Mesenquimatosas/citología , Proteína Homeótica Nanog , Proteínas del Tejido Nervioso/genética , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas de Unión al ARN/genética , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Proteínas de Dominio T Box/genética , Transducción Genética , alfa-Fetoproteínas/genética