Submitted to: Reproductive Biology
Publication Type: Abstract Only
Publication Acceptance Date: 1/5/2009
Publication Date: 2/3/2009
Citation: Singh, M., Amoah, E., Kannan, G., Stice, S., Donovan, D.M. 2009. Genome walk of an unknown upstream region of myostatin gene in Spanish goats. Reproductive Biology. Interpretive Summary: C. Problem— This work supports the effort to create knockout goats that lack expression from the myostatin gene. The creation of the knockout construct requires knowledge of the DNA sequence of ~5-7 Kbp of the goat myostatin gene. C. Accomplishment— This manuscript describes successes in subcloning goat genomic DNA fragments of the myostatin gene and identifying the clones harboring these goat genomic DNA vectors. C. Contribution of Accomplishment to Solving the Problem— This work moves the lab much closer to isolating the DNA, performing DNA sequence analysis, and compiling the goat myostatin gene for use in creating the knockout vector.
Technical Abstract: Myostatin (MSTN) gene product also known as growth differentiation factor (GDF8) is a member of the TGF-ß family of secreted proteins. It is shown to be a negative regulator of muscle mass development. Mutations in the MSTN gene have been reported in mice, cattle and humans that lead to muscular hypertrophy. Approaches directed towards inhibition of expression or/and disruption/deletion of the MSTN gene, therefore, have great potential in treatment of muscular dystrophy and in muscle wasting conditions such as cancer and AIDS in humans as well as in agricultural production systems. Goat meat has attained importance in the US not only among ethnic populations but also among mainstream consumers due to its nutritional value and lower fat content. A significant amount of goat meat is imported into the US from New Zealand and Australia. Enhancing goat meat production indigenously is, therefore, important. One of the potential approaches can be the production of animals with deleted/disrupted myostatin gene. For efficient gene disruption/deletion by gene-targeting, the length of the targeting flanking DNA region is important. Although sufficient nucleotide sequence of goat MSTN gene towards 3’ end of the coding sequence is available, only few hundred bases towards 5’ upstream region are known, which are insufficient for successful gene-targeting. The aim of this study, therefore, was to isolate and characterize upstream DNA fragment of sufficient length from the MSTN gene in Spanish goats which can be used as a targeting fragment in gene-targeting vectors. To achieve this goal we isolated genomic DNA from Spanish goats maintained at the Georgia Small Ruminant Research and Extension Center at Fort Valley State University and prepared 4 GenomeWalker libraries using GenomeWalker universal kit (Clonetac Labs.). A PCR-based DNA walking strategy was followed to isolate the unknown sequence upstream of the promoter region of goat MSTN gene. All 4 genomeWalker libraries were screened for PCR products using an adapter-specific sense and a MSTN gene-specific antisense primer set. Amplified products were confirmed and expanded by a “nested” PCR. Using this approach we have isolated PCR products ranging from 0.3 to 2.0 kb in all 4 libraries. Cloning and partial characterization of these amplified fragments will be presented.