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

A TAHERI-GHAHFAROKHI; M TAHMOORESPUR; H DEHGHANI; Y SHAMSHIRGARAN; H SUMER; J LIU; P J VERMA

doi:10.56093/ijans.v84i11.44725

Authors

A TAHERI-GHAHFAROKHI Monash Institute of Medical Research, Clayton VIC3168 Australia
M TAHMOORESPUR Monash Institute of Medical Research, Clayton VIC3168 Australia
H DEHGHANI Monash Institute of Medical Research, Clayton VIC3168 Australia
Y SHAMSHIRGARAN Ferdowsi University of Mashhad, Mashhad 91775–1163 Iran
H SUMER Swinburne University of Technology, Hawthorne, Australia
J LIU Monash Institute of Medical Research, Clayton VIC3168 Australia
P J VERMA Monash Institute of Medical Research, Clayton VIC3168 Australia

https://doi.org/10.56093/ijans.v84i11.44725

Keywords:

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

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.

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