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TALEN‐mediated gene targeting in porcine spermatogonia
Author(s) -
Tang Lin,
Bondareva Alla,
González Raquel,
RodriguezSosa Jose R.,
Carlson Daniel F.,
Webster Dennis,
Fahrenkrug Scott,
Dobrinski Ina
Publication year - 2018
Publication title -
molecular reproduction and development
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.745
H-Index - 105
eISSN - 1098-2795
pISSN - 1040-452X
DOI - 10.1002/mrd.22961
Subject(s) - biology , transcription activator like effector nuclease , nucleofection , germline , population , gene targeting , sox2 , germ cell , genetics , locus (genetics) , crispr , gene , spermatogenesis , microbiology and biotechnology , transfection , cas9 , embryonic stem cell , demography , sociology , endocrinology
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.