A 90-Day Safety Study of Meat from MSTN and FGF5 Double-Knockout Sheep in Wistar Rats.

Yue Zhao,Mingming Chen,Yao Li,Guang Yi,Kun Yu,Yan Li,Xueling Xu,Shiyu Qi,Shoulong Deng,Jinlong Zhang,Sujun Wu,Zhimei Liu,Xiaosheng Zhang,Zhengxing Lian,Xiaofei Guo

doi:10.3390/life12020204

Abstract

MSTN and FGF5 gene knockout sheep generated by the CRISPR/Cas9 system exhibit the ‘double-muscle’ phenotype, and increased density and length of hairs, providing valuable new breeding material. In a previous study, we obtained MSTN and FGF5 double-knockout sheep of significant breeding value. In this study, we carried out a 90-day feeding study in Wistar rats to assess the safety of genome-edited mutton. Seven rat groups with 10 females and 10 males per group were fed different concentrations (3.75%, 7.5%, and 15%) of double-knockout mutton or wild-type mutton in a conventional commercial diet for 90 days. At the end of the feeding, routine urine and blood tests and measurements of blood biochemical indicators were performed. Furthermore, the major organs of each group of rats were weighed and examined histopathologically. Although there were significant differences among the groups in some parameters, all values were within the normal ranges. Therefore, the 90-day rat feeding study showed that the meat from MSTN and FGF5 double-knockout sheep did not have any long-term adverse effects on rat health. This study also provides valuable reference information for assessing the safety of meat from animals with knockout of multiple genes.

Highlights

Myostatin (MSTN), called growth and differentiation factor-8 (GDF-8), is a member of the transforming growth factor-β (TGF-β) superfamily that negatively regulates skeletal muscle development and growth
In accordance with the National Standard for Food Safety 90-day oral toxicity test (Chinese Standard GB 15193.13-2015), a 90-day oral toxicity test using MSTN and Fibroblast growth factor 5 (FGF5) double-knockout lamb was performed on rats
The results showed no obvious signs of poisoning or toxicity-related deaths in all the groups, and there were no statistically significant differences in body weight per week, body weight gain, total food consumption, and urine among the groups

Summary

Introduction

Myostatin (MSTN), called growth and differentiation factor-8 (GDF-8), is a member of the transforming growth factor-β (TGF-β) superfamily that negatively regulates skeletal muscle development and growth. Various MSTN-KO animals (mouse, pig, sheep, and cattle) produced using genome editing techniques exhibit a distinct double-muscle phenotype [2,3,4,5]. Upon the use of genome editing technology to produce FGF5-KO goats and sheep, both groups of animals exhibited increased hair length and density [10,11]. Based on the role of MSTN with the FGF5 gene, we generated CRISPR/Cas9-mediated MSTN and FGF5 double-knockout sheep through microinjection. Both sgRNAs targeted the third exon of MSTN and FGF5 genes, resulting in a cysteine deletion and a frameshift mutation in genome edited sheep, respectively, which displayed an obvious ‘double-muscle’ phenotype and increased wool length phenotype. All F1 generation sheep were identified as showing a similar phenotype to the original generation, with genotypes MSTN+/− and FGF5+/−

Methods

Results

Discussion

Conclusion