Molecular reproduction and development, 2018-03, Vol.85 (3), p.250-261
2018
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Details
- Autor(en) / Beteiligte
- Titel
- TALEN‐mediated gene targeting in porcine spermatogonia
- Ist Teil von
- Molecular reproduction and development, 2018-03, Vol.85 (3), p.250-261
- Ort / Verlag
- United States: Wiley Subscription Services, Inc
- Erscheinungsjahr
- 2018
- Quelle
- Wiley-Blackwell Journals
- Beschreibungen/Notizen
- Spermatogonia represent a diploid germ cell population that includes spermatogonial stem cells. In this report, we describe new methods for isolation of highly enriched porcine spermatogonia based on light scatter properties, and for targeted mutagenesis in porcine spermatogonia using nucleofection and TALENs. We optimized a nucleofection protocol to deliver TALENs specifically targeting the DMD locus in porcine spermatogonia. We also validated specific sorting of porcine spermatogonia based on light scatter properties. We were able to obtain a highly enriched germ cell population with over 90% of cells being UCH‐L1 positive undifferentiated spermatogonia. After gene targeting in porcine spermatogonia, indel (insertion or deletion) mutations as a result of non‐homologous end joining (NHEJ) were detected in up to 18% of transfected cells. Our report demonstrates for the first time an approach to obtain a live cell population highly enriched in undifferentiated spermatogonia from immature porcine testes, and that gene targeting can be achieved in porcine spermatogonia which will enable germ line modification.
- Sprache
- Englisch
- Identifikatoren
- ISSN: 1040-452XeISSN: 1098-2795DOI: 10.1002/mrd.22961Titel-ID: cdi_pubmedcentral_primary_oai_pubmedcentral_nih_gov_6370346
- Format
- –
- Schlagworte
- Animals, Biotechnology, Clonal deletion, Gene deletion, gene editing, Gene Editing - veterinary, Gene targeting, Gene Targeting - veterinary, germ cells, Insertion, Light scattering, Male, Mutation, Non-homologous end joining, nuclease, nucleofection, pig, Site-directed mutagenesis, sorting, Spermatogenesis, Spermatogonia, Spermatogonia - cytology, Spermatogonia - metabolism, Stem cell transplantation, Stem cells, Swine, Testis - metabolism, Transcription activator-like effector nucleases, Transcription Activator-Like Effector Nucleases - genetics, Transcription Activator-Like Effector Nucleases - metabolism, Transfection