Performing Department
Animal & Avian Sciences
Non Technical Summary
Genetic gain in food animal production is critical for enhancing growth efficiency, animal health, and product quality. In pig production, genetic gain is achieved via selective breeding with desirable sires. At present, artificial insemination (AI) is a widely used option for exploiting this principle. However, the number of sperm that can be collected from an individual boar is a major limiting factor for widespread dissemination in the commercial pig production. Also, AI methodology limits the gains to the reproductive lifespan of a male, thereby resulting in the loss of superior genetic merit, and necessitating a new round of recruitment and identification of desirable sires. Thus, development of novel approaches for expanding the output and preservation of germline from desirable sires is of significant need. An overarching goal of this project is to generate lines of boar that completely lack endogenous spermatogonial stem cell (SSC), and use them as surrogates for exogenous SSC transplantation and spermatogenesis. To achieve this objective, we utilized the CRISPR/Cas9 system to edit the NANOS2 gene in pig embryos to generate offspring with mono-allelic and bi-allelic mutations. We found that NANOS2 knockout males lack germline but other aspects of testicular development are normal. In this study, the NANOS2-null animals will be propagated and exogenous SSC transplanted for initiation of spermatogenesis. This study will for the first time generate SSC-deficient animals by genome editing and test the feasibility of SSC transplantation in a large animal species. Arguably this project will have wide ranging applications in germline preservation technologies.
Animal Health Component
0%
Research Effort Categories
Basic
90%
Applied
10%
Developmental
(N/A)
Goals / Objectives
There are two major goals in this proposal: Aim-1. Generate a line of male pigs with testes that lack a germline but retain normal functions of somatic support cells. To achieve this, we have utilized recent advances in gene editing technologies (i.e., CRISPR/Cas9) to generate lines of pigs with an inactivate NANOS2 gene that is required for the survival of SSCs(1). We provide evidence that the biological role of NANOS2 is conserved in pigs, and that loss-of-function will result in elimination of the SSC population during fetal development. Importantly, this loss does not impact the functional capacity of somatic support cell populations.Aim-2. Determine the effectiveness of germline ablated pigs to serve as recipients for SSC transplantation. To achieve this, NANOS2 null male pigs will be transplanted with a cell suspension of undifferentiated spermatogonia isolated both from donor pig testes directly, and following a period of in vitro maintenance and expansion. Donor-derived spermatogenesis will then be assessed several months later. Also, the capacity of NANOS2 null recipient pigs to derive offspring with the donor haplotype via natural mating or AI will be evaluated. We anticipate that vast areas of donor-derived spermatogenesis will occur in recipient testes. Furthermore, the amount of sperm produced will be sufficient to generate offspring of the donor genotype similar to what has been achieved with SSC transplantation in germline ablated rodent models.
Project Methods
We will utilizethe site specific nucleases, specifically the CRISPR/Cas9 system to site specifically introduce double stranded breaks and ablate the NANOS2 gene in pig embryos to generate male offspring that s lack germline but other aspects of testicular development are normal. The NANOS2-null animals will be propagated and exogenous germ cells from donor boars will betransplanted for initiation of spermatogenesis. This study will, for the first time, generate germ cell-deficient animals by genome editing and test the feasibility of germ cell transplantation in a large animal species.