Germline complementation in sheep

Abstract

Controlling the male germline through natural mating and artificial insemination drives genetic improvement in sheep and cattle, respectively. Multiplication of the best male germline could be achieved at early developmental stages by using chimaeric embryos that comprise cells from distinct zygotic origins. However, their germline would also be chimaeric, restricting the rate of genetic gain since not all offspring would have the desired genotype. The work reported in this thesis provides a solution by knocking-out the RNA-binding proteins DAZL or NANOS2 in sheep, producing germ cell-deficient ‘host’ males, whose germ cells were then rescued by complementation with wild-type embryonic cells. CRISPR-Cas9 genome editing was used to disrupt DAZL or NANOS2 in male ovine fetal fibroblasts via homology-directed repair with insertion of premature termination codons and TaqI restriction sites. Cell strains were isolated by manual selection of mitotic cells, and those with homozygous editing were identified by PCR, sequencing, and TaqI digestion. Validated strains were used to generate embryos as nuclear donors for somatic cell cloning. Methodological improvements were made to the cloning procedure to prevent premature oocyte activation, which resulted in an approximately two-fold increase in blastocyst development. Testes sections from cloned DAZL-/- and NANOS2-/- neonates showed normal somatic support cells but lacked spermatogonia. Normal expression was observed for somatic cell-specific transcripts in the testes, confirming that the murine germ cell-deficient phenotype was conserved in sheep. Next, we tested if the vacant germ cell niche in NANOS2-/- hosts could be filled by making early embryonic chimaeras, using donor blastomeres expressing red fluorescent protein. The optimal protocol to form chimaeric blastocysts was by aggregating compacted morula embryos on day five. Overall, four lambs were generated from aggregation blastocysts. By analysing various tissues representative of the three germ layers with droplet digital PCR, the presence of cells from both origins was confirmed in the lamb from day five aggregation, and the others were found to be non-chimaeric. The chimaeric lamb had relatively even contribution of cell lineages across the body and an intact germline. This demonstrates that cells from wild-type donor embryos can fill the germline in genetically sterilised male hosts.