Abstract
In the present study, follistatin (FST) gene expression vectors with either a bicistronic gene transfer cassette alone, or a bicistron gene cassette carrying a matrix attachment region (MAR) were constructed and transfected to bovine fetal fibroblasts. Evaluations of both the integration and expression of exogenous FST indicated that the pMAR-CAG-FST-IRES-AcGFP1-polyA-MAR (pMAR-FST) vector had higher capacity to form mono- clonal transgenic cells than the vector without MAR, though transient transfection and integration efficiency were similar with either construct. Remarkably, protein expression in transgenic cells with the pMAR-FST vector was significantly higher than that from the bicistronic vector. Exogenous FST was expressed in all of the pMAR- FST transgenic mice at F0 ,F 1 and F2. Total muscle growth in F0 mice was significantly greater than in wild-type mice, with larger muscles in fore and hind limbs of transgenic mice. pMAR-FST transgenic mice were also found with more evenly distributed muscle bundles and thinner spaces between sarcolemma, which suggests a correlation between transgene expression-associated muscle develop- ment and the trend of muscle growth. In conclusion, a pMAR-FST vector, which excluded the resistant genes and frame structure, enhances and stabilizes FST gene expressions in both transfected cells and transgenic mice.
Highlights
Safety issues arising from genetically modified organisms (GMO) are of increasing concern, especially for commercialization of transgenic plants and animals
The results showed that FST-AcGFP1 had been integrated in all eight of the transgenic cell lines (Fig. 5) and that FST-matrix attachment region (MAR) had been integrated in seven of the eight transgenic cell lines (Fig. 5), which indicated that seven of the eight transgenic monoclonal cells had the pMAR-FST fully integrated into genome of host cells
The results showed that the exogenous genes were expressed in all of the pMAR-FST transgenic mice (Fig. 8)
Summary
Safety issues arising from genetically modified organisms (GMO) are of increasing concern, especially for commercialization of transgenic plants and animals. Linear DNA vectors without a backbone sequence, and selectable and resistant genes have not been used in transgenic animals. Its efficacy for transfection, integration and expression were first analyzed in transgenic cells This pMAR-FST vector was used to generate transgenic mice. PMAR-FST mice had skeletal muscles that were significantly enlarged when compared to control wild-type mice, suggesting that MAR sequence with FST bicistronic gene-transfer cassette was more appropriate for transgenic applications. To our knowledge, this is the first successful application of bovine MAR sequence along with FST and a bicistronic transfer cassette in transgenic animals
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