title: Production of CMAH Knockout Preimplantation Embryos Derived From Immortalized Porcine Cells Via TALE Nucleases.
abstract: Although noncancerous immortalized cell lines have been developed by introducing genes into human and murine somatic cells, such cell lines have not been available in large domesticated animals like pigs. For immortalizing porcine cells, primary porcine fetal fibroblasts were isolated and cultured using the human telomerase reverse transcriptase (hTERT) gene. After selecting cells with neomycin for 2 weeks, outgrowing colonized cells were picked up and subcultured for expansion. Immortalized cells were cultured for more than 9 months without changing their doubling time (~24 hours) or their diameter (< 20 µm) while control cells became replicatively senescent during the same period. Even a single cell expanded to confluence in 100 mm dishes. Furthermore, to knockout the CMAH gene, designed plasmids encoding a transcription activator-like effector nuclease (TALENs) pairs were transfected into the immortalized cells. Each single colony was analyzed by the mutation-sensitive T7 endonuclease I assay, fluorescent PCR, and dideoxy sequencing to obtain three independent clonal populations of cells that contained biallelic modifications. One CMAH knockout clone was chosen and used for somatic cell nuclear transfer. Cloned embryos developed to the blastocyst stage. In conclusion, we demonstrated that immortalized porcine fibroblasts were successfully established using the human hTERT gene, and the TALENs enabled biallelic gene disruptions in these immortalized cells.
authors.affiliation: Laboratory of Theriogenology and Biotechnology, Department of Veterinary Clinical Science, College of Veterinary Medicine and the Research Institute of Veterinary Science, Seoul National University, Gwanak-gu, Seoul, South Korea.
authors.forename: JoonHo
authors.initials: J
authors.lastname: Moon
authors.affiliation: Department of Chemistry, National Creative Initiatives Research Center for Genome Engineering, Seoul National University, Gwanak-gu, Seoul, South Korea.
authors.forename: Choongil
authors.initials: C
authors.lastname: Lee
authors.affiliation: Laboratory of Theriogenology and Biotechnology, Department of Veterinary Clinical Science, College of Veterinary Medicine and the Research Institute of Veterinary Science, Seoul National University, Gwanak-gu, Seoul, South Korea.
authors.forename: Su Jin
authors.initials: SJ
authors.lastname: Kim
authors.affiliation: Laboratory of Theriogenology and Biotechnology, Department of Veterinary Clinical Science, College of Veterinary Medicine and the Research Institute of Veterinary Science, Seoul National University, Gwanak-gu, Seoul, South Korea.
authors.forename: Ji-Yei
authors.initials: JY
authors.lastname: Choi
authors.affiliation: 1] Laboratory of Theriogenology and Biotechnology, Department of Veterinary Clinical Science, College of Veterinary Medicine and the Research Institute of Veterinary Science, Seoul National University, Gwanak-gu, Seoul, South Korea [2] Institute of Green Bio Science & Technology, Seoul National University, Pyeong Chang, Kangwon do, Korea.
authors.forename: Byeong Chun
authors.initials: BC
authors.lastname: Lee
authors.affiliation: Department of Chemistry, National Creative Initiatives Research Center for Genome Engineering, Seoul National University, Gwanak-gu, Seoul, South Korea.
authors.forename: Jin-Soo
authors.initials: JS
authors.lastname: Kim
authors.affiliation: 1] Laboratory of Theriogenology and Biotechnology, Department of Veterinary Clinical Science, College of Veterinary Medicine and the Research Institute of Veterinary Science, Seoul National University, Gwanak-gu, Seoul, South Korea [2] Institute of Green Bio Science & Technology, Seoul National University, Pyeong Chang, Kangwon do, Korea [3] Emergence Center for Food-Medicine Personalized Therapy System, Advanced Institutes of Convergence Technology, Seoul National University, Gyeonggi-do, Korea.
authors.forename: Goo
authors.initials: G
authors.lastname: Jang