Abstract

The chicken is a classic model organism that has provided key insights into embryonic development. Chicken embryos can be directly manipulated and observed during development while retaining the potential to reach adulthood. Despite this benefit, the utility of the chicken in studying development has been limited by the difficulty of introducing genetic changes to the genome. The recent development of cell culture conditions for chicken primordial germ cells (cPGC) has made it feasible to produce transgenic chickens, but there is still a lack of tools for introducing genetic modifications into cPGCs. Recombinase Mediated Cassette Exchange (RMCE) is a technique that has been utilized in traditional genetic systems to generate multiple alleles at a given locus but has not yet been adapted to the chicken. In order to use RMCE in the chicken, we inserted Lox sites into cPGC using CRISPR/Cas9. We targeted the ovalbumin locus and potential genomic safe harbor sites (GSH) identified using genomic data. We performed RMCE to exchange green fluorescent protein (GFP) into these loci. We observed RMCE efficiency as less than 1% at each loci. We then designed a system using a drug inducible Caspase 9 (iCasp9) to select for cells that underwent cassette exchange. This method enabled us to obtain a population of 100% edited cells. We anticipate that this tool will increase the utility of the chicken as a model organism, livestock, and bioreactor.

Degree

College and Department

Life Sciences

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