Establishment of an Efficient Immortalization Strategy Using HMEJ-Based bTERT Insertion for Bovine Cells

Area of Science:

• Cell Biology
• Genetics
• Animal Science

Background:

• Immortalized cell lines are crucial for studying immune disorders and cellular metabolism due to their stable characteristics.
• Current methods for immortalizing bovine cells, primarily using human telomerase reverse transcriptase (hTERT), are inefficient and unreliable.
• A lack of suitable immortalized bovine cell models limits research into bovine molecular functions and signaling pathways.

Purpose of the Study:

• To identify a potent biofactor for immortalizing bovine cells with advantages over hTERT.
• To establish an efficient and manageable strategy for immortalizing bovine primary cells.
• To generate stable immortalized bovine fetal fibroblast cell lines.

Main Methods:

• Identified bovine telomerase reverse transcriptase (bTERT) as a novel immortalization biofactor.
• Developed a homology-mediated end-joining strategy for bTERT insertion at the ROSA26 locus.
• Generated immortalized bovine fetal fibroblast cell lines.

Main Results:

• Successfully generated immortalized bovine fetal fibroblast cell lines with stable characteristics.
• Demonstrated that bTERT offers advantages over hTERT for bovine cell immortalization.
• Identified post-translational modification of bTERT as a limitation in immortalizing bovine bone marrow-derived macrophages.

Conclusions:

• Developed an innovative and efficient method for high-quality immortalization of bovine primary cells.
• The new strategy expands the utility of immortalized bovine model cell lines for future research.
• Overcame limitations of existing methods, paving the way for advanced studies in bovine biology.