title: Robust method for TALEN-edited correction of pF508del in patient-specific induced pluripotent stem cells.
abstract: Cystic fibrosis is one of the most frequent inherited rare diseases, caused by mutations in the cystic fibrosis transmembrane conductance regulator gene. Apart from symptomatic treatments, therapeutic protocols for curing the disease have not yet been established. The regeneration of genetically corrected, disease-free epithelia in cystic fibrosis patients is envisioned by designing a stem cell/genetic therapy in which patient-derived pluripotent stem cells are genetically corrected, from which target tissues are derived. In this framework, we present an efficient method for seamless correction of pF508del mutation in patient-specific induced pluripotent stem cells by gene edited homologous recombination. Gene edition has been performed by transcription activator-like effector nucleases and a homologous recombination donor vector which contains a PiggyBac transposon-based double selectable marker cassette.This new method has been designed to partially avoid xenobiotics from the culture system, improve cell culture efficiency and genome stability by using a robust culture system method, and optimize timings. Overall, once the pluripotent cells have been amplified for the first nucleofection, the procedure can be completed in 69 days, and can be easily adapted to edit and change any gene of interest.
authors: Caubet-Cimera Foundation, Hospital Joan March, Ctra Soller Km 12, 07110, Bunyola, Mallorca, Spain. telomerasi@yahoo.es.
authors: María Vicenta
authors: MV
authors: Camarasa
authors: Caubet-Cimera Foundation, Hospital Joan March, Ctra Soller Km 12, 07110, Bunyola, Mallorca, Spain.
authors: Víctor Miguel
authors: VM
authors: Gálvez
mesh_terms: D001483:Base Sequence; D018920:Coculture Techniques; D055028:Comparative Genomic Hybridization; D003550:Cystic Fibrosis / Q000235:genetics / Q000628:therapy*; D017931:DNA Primers / Q000235:genetics; D004251:DNA Transposable Elements; D003851:Deoxyribonucleases / Q000235:genetics*; D061252:Feeder Cells; D005818:Genetic Engineering; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000502:physiology*; D020014:K562 Cells; D059767:Recombinational DNA Repair; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion; D014162:Transfection
chemical_list: D017931:DNA Primers; D004251:DNA Transposable Elements; D003851:Deoxyribonucleases