Efficient TALEN-mediated mutagenesis on a highly conserved region of myostatin gene in mouse embryonic stem cells

Abstract

Myostatin is a negative regulator of muscle growth. The naturally occurring mutations of myostatin gene (MSTN) are strongly associated with meat production traits in livestock. Thus far, many attempts have been made to knock- down/knock-out MSTN across numerous species. The main objective of this study was to construct and evaluate the efficiency of a single pair of transcription activator like effectors nucleases (TALENs), to target MSTN in mouse. A target site in a highly conserved region of MSTN exon 2, with minimal off-target counts was selected for targeting by customized TALENs. The targeted site was identified similarly among mouse, cattle, sheep, goat and pig. A modified Golden Gate TALEN generating platform was used for producing the myostatin specific TALEN- expressing plasmids. Generated myostatin TALENs were evaluated in mouse embryonic stem cells (mESC) using high resolution melting (HRM) analysis for detecting newly arising TALEN-induced mutations in the myostatin gene. Our results showed that generated TALENs are functional and able to disrupt the myostatin gene at an efficiency of up to 35% in mESC. If translated to livestock species, this approach can be utilized for producing myostatin modified animals with lower food conversion ratios. The precision and efficiency achieved may even enable direct targeting of zygotes with TALENs, affecting a step-change in the generation of knock-out livestock.

Keywords

• Gene targeting
• Golden-Gate cloning
• High resolution melting
• Myostatin
• TALENs