Targeted Nucleotide Exchange in Bovine Myostatin Gene

• Published in: Animal biotechnology 2009-01, Vol.20 (1-4), p.15-27
• Main Authors: Grisolia, A.B, Curi, R.A, Lima, V.F.M. de, Olmedo, H. Parekh, Kmiec, E, Nunes, C.M, Aoki, S.M, Garcia, Jose Fernando
• Format: Article
• Language: English
• Subjects: amino acid substitution; Animals; Base Sequence; cattle; Cattle - genetics; cattle breeds; Cell Differentiation; Cell Line; DNA, Single-Stranded - genetics; Female; Gene Expression Regulation; genes; Genetic Engineering; genetic polymorphism; genetic transformation; growth differentiation factor 8; growth differentiation factors; Molecular Sequence Data; Mutation; Myoblasts; Myoblasts - cytology; Myoblasts - metabolism; Myostatin; Myostatin - genetics; Myostatin - metabolism; Nellore; oligonucleotides; Piedmontese; Point Mutation; Polymorphism, Genetic; RNA - genetics; single-stranded DNA; site-directed mutagenesis; stem cells; Transfection
• ISSN: 1049-5398; 1532-2378
• DOI: 10.1080/10495390802594693

The myostatin gene, known as Growth Differentiation Factor 8 (GDF8), located at chromosome 2 (BTA2) in cattle, is specifically expressed during embryo development and in the adult skeletal muscle. Molecular analysis of this gene reveals the presence of three exons and two introns. Several cattle breeds, such as Piedmontese, Belgian Blue, Blond'Aquitaine, among others, show polymorphisms in this gene, which are directly related to double muscling phenotype. Piedmontese cattle shows a nucleotide transition G → A (G938A) at exon 3, resulting in the substitution of cysteine to tyrosine, leading to a protein structure change, which determines myostatin inactivation and consequent muscular hypertrophy. The objective of this work was to implant the polymorphism G938A, naturally existent in Piedmontese breed, into in vitro propagated foetal myoblasts, from Nellore cattle. Single strand DNA (ssDNA) oligonucleotides were used to direct the same nucleotidic transition (G938A) to exon 3. Two transfection protocols (cationic lipid solution and electroporation) were tested and, 48 hours after transfection, RNA and DNA were extracted from myoblasts. Reverse transcription and polymerase chain reaction (PCR) were performed, using primers flanking the mutation region. The PCR products were cloned and analyzed by DNA sequencing, and it was possible to detect the nucleotidic C→T transition at position 938, in the electroporated myoblasts. The existence of a positive signal in the transfection indicates that ssDNA oligonucleotides can be used to introduce this point mutation in specific functional gene sites.