Male fertility restored by transplanting primordial germ cells into testes: a new way towards efficient transgenesis in chicken

Abstract

The ongoing progress in primordial germ cell derivation and cultivation is opening new ways in reproductive biotechnology. This study tested whether functional sperm cells can be matured from genetically manipulated primordial germ cells after transplantation in adult testes and used to restore fertility. We show that spermatogenesis can be restored after mCherry-expressing or GFP-expressing primordial germ cells are transplantated into the testes of sterilized G0 roosters and that mCherry-positive or GFP-positive non-chimeric transgenic G1 offspring can be efficiently produced. Compared with the existing approaches to primordial germ cell replacement, this new technique eliminates the germ line chimerism of G0 roosters and is, therefore, faster, more efficient and requires fewer animals. Furthermore, this is the only animal model, where the fate of primordial germ cells in infertile recipients can be studied.

Resources for genome editing in livestock: Cas9-expressing chickens and pigs

“…In this study, EGFP-PGCs were derived from the germinal crescent as previously described (50). They were maintained in culture and stably transfected with a Cas9 construct which was mediated by phiC31 integrase as previously described (51). Briefly, 5 × 10 6 of EGFP-PGCs were resuspended in a total volume of 100 μl Nucleofector Solution V (Lonza, Switzerland) premixed with 10 μg of Cas9 plasmid and the same amount of phiC31 integrase.…”

Applications of Gene Editing in Chickens: A New Era Is on the Horizon

“…Since PGCs are precursors of sperm, researchers suggested that roosters could be used as recipient for exogenous transfer of genetically-modified PGCs which may improve the germline transmission rate (Trefil et al, 2017 ). Chicken embryos and adult roosters were chemically or physically sterilized to create a surrogate for external PGC donors (Trefil et al, 2006 ; Nakamura et al, 2008 , 2010 ; Ghadimi et al, 2017 ).…”

“…Performing injection of donor spermatogonial cells led to reestablishment of male function in 50% of the roosters only 5 weeks after injection (Trefil et al, 2006 ). Spermatogenesis was restored 4 weeks later in the case of PGC-transplantation compared to spermatogonial cells; however, PGCs exhibited higher efficiency in repopulating the seminiferous epithelium (Trefil et al, 2006 , 2017 ). This was very beneficial in the case of transplantation of genetically modified PGCs into mature roosters after complete irradiation (Trefil et al, 2017 ).…”

“…Spermatogenesis was restored 4 weeks later in the case of PGC-transplantation compared to spermatogonial cells; however, PGCs exhibited higher efficiency in repopulating the seminiferous epithelium (Trefil et al, 2006 , 2017 ). This was very beneficial in the case of transplantation of genetically modified PGCs into mature roosters after complete irradiation (Trefil et al, 2017 ). Male fertility was reestablished after the transplantation of GFP- or mCherry-expressing PGCs and resulted in almost 100% germline transmission (Trefil et al, 2017 ).…”

“…This was very beneficial in the case of transplantation of genetically modified PGCs into mature roosters after complete irradiation (Trefil et al, 2017 ). Male fertility was reestablished after the transplantation of GFP- or mCherry-expressing PGCs and resulted in almost 100% germline transmission (Trefil et al, 2017 ). The prominent advantage of this method is the certainty of the germline transmission and the low number of animals used in the experiment; hence reducing time and costs for testing high number of chimeric roosters.…”