Abstract
BackgroundThe production of transgenic chicken cells holds great promise for several diverse areas, including developmental biology and biomedical research. To this end, site-specific gene integration has been an attractive strategy for generating transgenic chicken cell lines and has been successfully adopted for inserting desired genes and regulating specific gene expression patterns. However, optimization of this method is essential for improving the efficiency of genome modification in this species.ResultsHere we compare gene knock-in methods based on homology-independent targeted integration (HITI), homology-directed repair (HDR) and homology mediated end joining (HMEJ) coupled with a clustered regularly interspaced short palindromic repeat associated protein 9 (CRISPR/Cas9) gene editing system in chicken DF-1 cells and primordial germ cells (PGCs). HMEJ was found to be a robust and efficient method for gene knock-in in chicken PGCs. Using this method, we successfully labeled the germ cell specific gene DAZL and the pluripotency-related gene Pou5f3 in chicken PGCs through the insertion of a fluorescent protein in the frame at the 3′ end of the gene, allowing us to track cell migration in the embryonic gonad. HMEJ strategy was also successfully used in Ovalbumin, which accounts for more than 60% of proteins in chicken eggs, suggested its good promise for the mass production of protein with pharmaceutical importance using the chicken oviduct system.ConclusionsTaken together, these results demonstrate that HMEJ efficiently mediates site-specific gene integration in chicken PGCs, which holds great potential for the biopharmaceutical engineering of chicken cells.
Highlights
Gene modification technologies utilized in chicken have great potential for applications in fields such as developmental biology and biomedical research
Our research demonstrated that homology mediated end joining (HMEJ) was a robust and efficient method for gene knock-in in chicken primordial germ cells (PGCs)
CRISPR/Cas9 induces targeted DNA cleavage in chicken cells The DAZL gene in DF-1 cells was targeted to determine the efficiency of the CRISPR/Cas9 system in chicken
Summary
Gene modification technologies utilized in chicken have great potential for applications in fields such as developmental biology and biomedical research. The production of protein “drugs” via the use of transgenic animals is an emerging field for the pharmaceutical industry This requires the integration of a desired gene specific to a protein of interest into the genome of recipient animals, making its economically significant expression inherited in successive generations of animals [1]. The production of transgenic chicken cells holds great promise for several diverse areas, including developmental biology and biomedical research To this end, site-specific gene integration has been an attractive strategy for generating transgenic chicken cell lines and has been successfully adopted for inserting desired genes and regulating specific gene expression patterns. Site-specific gene integration has been an attractive strategy for generating transgenic chicken cell lines and has been successfully adopted for inserting desired genes and regulating specific gene expression patterns Optimization of this method is essential for improving the efficiency of genome modification in this species
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