TALEN‐mediated gene targeting in porcine spermatogonia. Issue 3 (22nd February 2018)

• Record Type: Journal Article
• Authors: Tang, Lin; Bondareva, Alla; González, Raquel; Rodriguez‐Sosa, Jose R.; Carlson, Daniel F.; Webster, Dennis; Fahrenkrug, Scott; Dobrinski, Ina
• Is Part Of: Molecular reproduction and development. Volume 85:Issue 3(2018)
• Page Start: 250
• Page End: 261
• Publication Date: 2018-02-22
• Subjects: gene editing -- germ cells -- nuclease -- nucleofection -- pig -- sorting; Reproduction -- Periodicals; Molecular biology -- Periodicals; Molecular genetics -- Periodicals; Embryology -- Periodicals; 571.8
• DOI: 10.1002/mrd.22961
• Languages: English
• ISSNs: 1040-452X

Abstract

Abstract : 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.