Crosstalk between myostatin and callipyge in CRISPR/Cas9-edited goat fibroblast cells
Helia Fathpour1, Mansooreh Fouladi2, Farnoosh Jafarpour3
1ACECR Institute of Higher Education (Isfahan Branch), Iran; Department of Medical Microbiology & Infectious DiseasesUniversity of Manitoba, Winnipeg, MB, Canada.
Abstract:
Myostatin (MSTN) and Callipyge (CLPG) genes are key regulators of muscle growth. While MSTN inhibits muscle development, the CLPG mutation induces muscle hypertrophy through a specific imprinted genetic mechanism. The interaction between these genes remains of interest for improving livestock muscle traits. In this study, CRISPR/Cas9 was employed to edit MSTN and CLPG genes in goat fibroblast cells via electrotransfection. Cells were selected using puromycin antibiotic, and gene-editing efficiency was evaluated through Sanger sequencing. Gene expression changes were analyzed using RT-qPCR analysis. MSTN gene knockout resulted in significant downregulation of MSTN and CLPG, while GTL2 expression was upregulated by more than 50-fold. Additionally, myosin heavy chain genes (MYH1, MYH3, MYH4) were strongly upregulated, with MYH3 13-fold and MYH4 30-fold increase in the expression. In CLPG-edited cells, the expression of MSTN, TRIM28, and CLPG was reduced, while GTL2 was upregulated by 6-fold. MYH3 and MYH4 expression increased 4-fold in CLPG-edited cells, though the increase was less pronounced compared to MSTN-edited cells. DLK1 expression was undetectable in both non-edited control and gene-edited fibroblast cells. Our findings support the interaction between MSTN and CLPG, contributing to the regulation of muscle growth. Notably, the study also highlights the challenges associated with editing imprinted genes like CLPG and suggests that TRIM28 may play a role downstream of CLPG regulation. These results provide valuable insights into muscle development regulation, offering potential applications in livestock genetic improvement.
Insights
Gene editing of Myostatin (MSTN) and Callipyge (CLPG) in goats reveals their interaction in muscle growth regulation. MSTN editing significantly boosted muscle development genes, while CLPG editing showed a less pronounced effect.
Area of Science:
- Animal Genetics
- Molecular Biology
- Biotechnology
Background:
- Myostatin (MSTN) inhibits muscle growth, while the Callipyge (CLPG) mutation promotes muscle hypertrophy via genetic imprinting.
- Understanding the interplay between MSTN and CLPG is crucial for enhancing muscle traits in livestock.
Purpose of the Study:
- To investigate the interaction between MSTN and CLPG genes using CRISPR/Cas9 gene editing in goat fibroblast cells.
- To analyze the effects of MSTN and CLPG gene editing on muscle-specific gene expression.
Main Methods:
- CRISPR/Cas9 gene editing was used to modify MSTN and CLPG genes in goat fibroblasts.
- Gene editing efficiency was confirmed using Sanger sequencing.
- Gene expression levels were quantified via RT-qPCR analysis.
Main Results:
- MSTN gene knockout led to significant upregulation of myosin heavy chain genes (MYH3, MYH4) and GTL2, with downregulation of MSTN and CLPG.
- Editing the CLPG gene resulted in increased expression of GTL2 and myosin heavy chain genes (MYH3, MYH4), alongside reduced MSTN and TRIM28 expression.
- DLK1 expression was not detected in either control or edited cells.
Conclusions:
- The study confirms an interaction between MSTN and CLPG in regulating muscle growth.
- Editing imprinted genes like CLPG presents challenges, and TRIM28 may be involved in CLPG's regulatory pathway.
- Findings offer insights into muscle development and potential strategies for livestock genetic improvement.
