HMEJ-mediated efficient site-specific gene integration in chicken cells

Area of Science:

• Genetics and Molecular Biology
• Developmental Biology
• Biotechnology

Background:

• Transgenic chicken cells are valuable for developmental biology and biomedical research.
• Site-specific gene integration is key for creating transgenic chicken cell lines.
• Optimizing genome modification efficiency in chickens is crucial.

Purpose of the Study:

• To compare gene knock-in efficiencies of HITI, HDR, and HMEJ methods using CRISPR/Cas9 in chicken cells.
• To evaluate the suitability of HMEJ for gene editing in chicken primordial germ cells (PGCs).
• To explore the potential of HMEJ for biopharmaceutical applications in chickens.

Main Methods:

• Utilized clustered regularly interspaced short palindromic repeat associated protein 9 (CRISPR/Cas9) gene editing.
• Compared homology-independent targeted integration (HITI), homology-directed repair (HDR), and homology mediated end joining (HMEJ) for gene knock-in.
• Applied HMEJ in chicken DF-1 cells and PGCs, targeting genes like DAZL, Pou5f3, and Ovalbumin.

Main Results:

• Homology mediated end joining (HMEJ) demonstrated robust and efficient gene knock-in in chicken PGCs.
• Successfully labeled germ cell specific gene DAZL and pluripotency gene Pou5f3 in chicken PGCs using HMEJ for cell migration tracking.
• Demonstrated HMEJ applicability for Ovalbumin gene editing, indicating potential for pharmaceutical protein production in chicken oviducts.

Conclusions:

• HMEJ is a highly efficient method for site-specific gene integration in chicken PGCs.
• This approach shows significant promise for advancing biopharmaceutical engineering using chicken cells.
• HMEJ facilitates genetic manipulation for research and biotechnological applications in avian species.