A Rapid, Non-Invasive Blood-Based Reporter System for Early Selection of High-Percentage Germline Chimeras in Transgenic Chicken Production

The production of genetically modified chickens via primordial germ cells (PGC) transplantation is hindered by the lack of efficient methods to assess germline chimerism in G0 founders. Currently, researchers face a binary choice between destructive sampling and prolonged blind rearing, as no non-invasive screening tool exists to assess chimerism prior to maturity. Conventional approaches rely on either invasive gonadal biopsy or time-consuming progeny testing, both of which significantly delaying breeding timelines and increasing costs. Here, we present a transformative, non-invasive screening strategy that enables longitudinal, real-time quantification of exogenous PGC engraftment. We generated male chicken PGC co-expressing a secreted Gaussian luciferase (Gluc) reporter and GFP via piggyBac transposition. Upon microinjection into recipient embryos, these PGC successfully colonized the gonads and gave rise to germline-competent chimeras. Gluc activity in peripheral blood was positively associated with donor PGC' presence in testes, allowing for discrimination between male and female chimeras and revealing individual variation in chimerism degree. This secreted reporter system facilitated the identification of high-percentage male chimera pre-sexual maturity, with sustained or increased signal at 120 days, confirming stable transgene expression. Progeny testing demonstrated that higher blood Gluc activity was associated with greater germline transmission efficiency (75–81%). Moreover, Gluc-based screening detected more transgene-positive offspring than GFP fluorescence and confirmed stable inheritance of the reporter in G1 progeny. By enabling early, non-destructive selection of optimal breeding founders, our method drastically reduces housing costs, resource use, and the time required to establish transgenic chicken lines. Collectively, these results demonstrate that blood Gluc activity could be a quantitative surrogate marker of donor PGC colonization and provides a rapid, non-invasive method for selection of high-percentage germline chimeras prior to sexual maturity in transgenic chicken production, thereby reducing housing costs and accelerating transgenic chicken line establishment.