Optimization and testing of new methods for the generation of zebra finch transgenics
Sensory and Behavioural Biology
Final Report Abstract
Young songbirds, similar to human infants learning to speak, acquire their songs through vocal imitation of adult conspecifics. Due to these shared mechanisms, songbirds are widely used as an animal model for studying the molecular, neural, and physiological aspects of vocal production learning. However, the ability to reliably and routinely perform germline modifications in songbirds is still lacking. Since 2009, only four laboratories have successfully generated transgenic finches, resulting in a total of six transgenic lines. The efficiency of generating these lines has been low, ranging from 1% to 10%. Most transgenic zebra finches have been produced through lentiviral injection into freshly laid eggs at Stage X, targeting primordial germ cells (PGCs). In a publication from 2020, PGCs were extracted from the blood of approximately 3-day-old zebra finch embryos, cultured, infected with a lentivirus containing an ubiquitous promoter driving green fluorescence protein (GFP), and reinjected into a Stage X embryo. This method showed good efficiency. All existing methods for generating transgenic zebra finches involve the production of a chimeric F0 generation, which then needs to be crossed with wild-type individuals to generate transgenic F1 offspring. Previous publications have reported low efficiency (~13%) in generating transgenic F1 offspring, except for the 2020 study, which reported higher efficiency. The first transgenic chicken was produced in 1986 using a method similar to the one employed for the first transgenic zebra finch in 2009. Since then, several different and more efficient methods for generating transgenic chickens and quails have been developed. Therefore, we proposed to compare these established methods in chickens and quails and adapt them for generating transgenic zebra finch lines. In pursuit of this aim, we have tested five published approaches to generate transgenic zebra finches, but without final success. 1) lentivirus injection at Stage X and Stage 16-18 resulted in improved hatching rates but no transgenic individuals 2) sperm transfection-assisted gene editing (STAGE) resulted in obtaining, maintaining and artificially inseminating sperm, but manipulation of sperm was unsuccessful; 3) in vivo electroporation of zebra finch testis resulted in long-lasting expression of the inserted gene and fertile males but no transgene transmission to the next generation; 4) the method reported as highly efficient in generating transgenic zebra finches (Gessara et al., 2020) could not be replicated by us to produce transgenic F1 offspring; 5) cultivating PGCs extracted from the gonads of 6-day-old zebra finches for three months, which is three times longer than previously reported for zebra finches. We are currently attempting to manipulate these cultured PGCs. Modifying the genome of songbirds continues to be a benchmark that needs to be achieved to fully harness this species’ potential as an animal model for vocal production learning, as well as social and cognitive behavior. During the past three years we have made significant progess in identifying specific hurdles and solutions, but not yet been able to establish a reliable pipeline.
