Sequencing and Bioinformatic Investigation of Introducing a Repressive Micro-RNA Target Sites in the 3'UTR of Myostatin Gene in some Indigenous Sheep Breeds of Iran

Myostatin, known as the growth and differentiation factor 8 (GDF8), is a negative regulator of skeletal muscle growth in mammals. This gene is conserved across mammalian species and expressed in developing as well as mature skeletal muscles fiber cells. It has been demonstrated that a natural mutation in myostatin (MSTN) gene is effective in muscle tissue overgrowth in species such as cattle, sheep, pig and mouse. Later on, with administration of genome editing techniques on rabbit (17), pig (16) and dog (31), double muscling trait was successfully achieved by modifying MSTN gene. It can be inferred from the studies that the removal of MSTN’s inhibitory role will leads to muscle increase, an observation similar to that of other mammalian species. Therefore, it can be considered as a candidate gene for growth and carcass traits. This simple mutation can be used as a model for genetic engineering of farm animals to improve growth traits. In total, blood samples of 15 sheep from each breed of Dalagh, Baluchi and Zel were collected. Genomic DNA was extracted from whole blood using Guanidium Thiocyanate-Silica Gel method (Diatom DNA Prep. 100, Isogene, Russia) following the manufacturer’s instruction. The integrity of the extracted DNA was assessed by electrophoresis on a 0.8% agarose gel and the purity of the obtained DNA was evaluated by Epoch microplate spectrophotometer (BioTek, USA). A 2180 bp region from 3UTR of ovine MSTN gene was amplified by standard PCR reaction in a total volume of 25 µl. Tree sets of specific overlapping primers were used to amplify part of the 3UTR region in MSTN gene (Table 1). The PCR products then purified by ethanol precipitation method (14) and sequenced. The sequencing results homology were checked by BLAST and assembled using CLC sequence viewer 8.0. Then miRNA target sites were analyzed to create a potent in silico modification which serve as a target site for the microRNAs miR-1 and miR-206 with suppressing effect on MSTN transcript. At the end to assess the formation of any new and undesired motif due to the creation of our in silico modification, the whole area analyzed with the motif finder application. This study was performed to identify and compare DNA sequence of a 2180 bp region from the 3'UTR of myostatin gene in Dalagh, Baluchi and Zel sheep breeds and with the aim of introducing an in silico modification to introduce a mutation with a positive impact on growth rate. Results showed that there was a high similarity between 3' UTR sequences of GDF8 gene in Zel, Dalagh and Baluchi sheep breeds. All samples were monomorphic and had the g+6723G allele, which do not cause double muscle phenotype (Figure 2). An in silico approach employed to modify the 3'UTR of the myostatin gene in this indigenous sheep breeds in order to create miR-1 and miR-206 (ACATTCCA) target sequences naturally occurring in the Texel sheep (Figure 3). After applying these changes, the possibility of creating unwanted new regulatory elements was investigated using the motif finder software. The results showed that the introduced mutations did not create any new motifs that had a known regulatory role in mammals. It was demonstrated that this mutation can attribute to 38% of the additive genetic variance for muscle depth in the Charollais lambs (12). In another study, this mutation found to have a significant increase in muscle mass and reduced carcass fat in Norwegian White sheep (6). Therefore, this single modification can be considered as the best mutation for double muscling due to its large effect on the muscling phenotype. Due to the large effect that g+6723G>A mutation has on the phenotype double muscle and also the absence of other known effects on the phenotypes, this mutation could be considered as one of the best candidates for genome editing that can create indigenous sheep with overgrown muscle phenotype in the future by using of genetic engineering techniques. It is feasible to introduce this mutated allele by genetic engineering methods as a desirable genetic modification for improving indigenous sheep breeds. Advantages of using this approach include increasing the genetic progress of breeding programs in compare to traditional methods and maintaining the environmental compatibility of indigenous sheep breeds.
Muscle tissue, Sheep, Myostatin, Micro RNA