Document Type : Research Paper
Authors
1 1. Department of Animal Science, Faculty of Animal and Food Science, Agricultural Sciences and Natural Resources University of Khuzestan,Ahvaz, Iran. 2. Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, T
2 1. Department of Animal Science, Faculty of Animal and Food Science, Agricultural Sciences and Natural Resources University of Khuzestan,Ahvaz, Iran.
3 Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
4 Department of Medical Biotechnology School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran;
Abstract
The myostatin gene (MSTN) plays an important role in the regulation of skeletal muscle mass, and the translational inhibition of this gene indicates an increase in muscle mass, which is known as the "double-muscle phenotype." Disruption of MSTN gene expression using CRISPR/Cas9 genome editing technology has shown muscle growth and growth rate in livestock species including sheep and goats. This research was carried out in order to produce Vramini sheep embryos carrying MSTN gene knockout and to examine the embryos. In the first step, CRISPOR software (http://crispor.tefor.net/) was used for initial screening to design and select guide RNA. Two guide RNAs targeting an exon of the MSTN gene were selected. Then, 50 ng of the mixture of two guide RNAs along with Cas9 mRNA was microinjected into the oocytes at the same time. Among the 12 embryos produced, five embryos showed CRISPR technology markers. Sequencing results showed two types of mutations for guide RNA1 and guide RNA2 separately, but both types of guide RNA did not function simultaneously in any of the embryos. The percentage of cell cut with Synthego site was reported as 83%. The nucleotide sequence of the exon of a Varamini sheep MSTN was different from the reference sequence registered in NCBI. The amino acid sequence of the embryos carrying the MSTN gene knockout showed the termination code, which can cause a change in its protein synthesis, followed by an increase in muscle volume.
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