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Production of CMAH Knockout Preimplantation Embryos Derived From Immortalized Porcine Cells Via TALE Nucleases

Cited 1 time in Web of Science Cited 2 time in Scopus
Authors
Moon, JoonHo; Lee, Choongil; Kim, Su Jin; Choi, Ji-Yei; Lee, Byeong-Chun; Kim, Jin-Soo; Jang, Goo
Issue Date
2014-05
Citation
Molecular Therapy - Nucleic Acids, Vol.3, p. e166
Keywords
CMAH knockoutimmortalizationsomatic cell nuclear transfertranscription activator-like effector nuclease
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 (similar to 24 hours) or their diameter (< 20 mu m) 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.
ISSN
2162-2531
URI
http://hdl.handle.net/10371/165643
DOI
https://doi.org/10.1038/mtna.2014.15
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