Creation of transgenic chicken for production of human therapeutic proteins in ovo

Hyman, Sophia Yekara
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The transgenic chicken in ovo expression platform for production of pharmaceutical proteins, presents a more cost-effective, high-yielding alternative to current cell-based production systems. The aim in this project was to contribute towards developing a transgenic chicken bioreactor that will ultimately express high levels of therapeutic protein, Tenecteplase, in place of endogenous egg white proteins Ovalbumin and Ovomucoid in ovo. The gene-editing approach was CRISPR-Cas9 gene drive constructs in isolated chicken germ cells. The Ovalbumin- and Ovomucoid-specific gene drive constructs were generated through multiple PCR, restriction digests and cloning procedures. Chicken PGCs, extracted from blood of stage 14-17HH embryos, were propagated in vitro upon chicken embryonic fibroblast feeder cells. PGCs exhibited typical spherical morphology and stained positive for germ cell surface marker SSEA-1 in fluorescence immunocytochemistry. Unfortunately, gene drive constructs failed to produce desired transgene integration following transfection and antibiotic selection of PGCs. In busulfan-based sterilisation, endogenous PGCs were reduced significantly by 70% using 1.5μg/μl busulfan (P<0.05). Donor PGCs injected into day 3 embryonic vasculature were not detected in gonads of previously sterilised embryos at day 6 incubation; likely due to the sup- optimal fluorescent-vector-based tracking of donor PGCs or injection via unreliable mouth- controlled pipette. Future work must investigate manipulating the various factors influencing gene editing at target loci, including cell conditions and pathways favouring HDR, accessible chromatin states for CRISPR-Cas9 machinery, gRNA design, and donor gene drive template conformation. These preliminary experiments, towards production of a transgenic chicken bioreactor, have provided valuable resources and insight for achievement of this pursuit.
A dissertation submitted in fulfilment of the requirements for the degree of Master of Science to the Faculty of Science,School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg, 2023