Integration, stability and expression of the E. coli phytase transgene in the Cassie line of Yorkshire Enviropig™.

The genomic structure and generational stability of the transgene carried by the Cassie (CA) line of the transgenic Enviropig™, a prospective food animal, are reported here. This transgene is composed of the Escherichia coli phytase coding sequence regulated by the mouse parotid secretory protein promoter to direct secretion of phytase in the saliva. In the CA line the transgene integrated in chromosome 4 is present as a concatemer of three copies, two in a head to tail orientation and the third in a reverse orientation 3' to the other copies with a 6 kbp deletion in the 5' promoter region. The overall size of the integrated transgene complex is 46 kbp. During integration a 66 kbp segment of the chromosome was deleted, but a BLAST search of the segment from a GenBank clone did not reveal any essential genes. The transgene integration site was stable through 9 generations analyzed. Phytase activity in the saliva was similar among 11 day old hemizygous boars and gilts and remained relatively constant through nine generations of hemizygous pigs. However, as the pigs grew there generally was a gradual decrease in activity that stabilized when pigs reached the finisher phase of growth (4-6 months old). Homozygous pigs exhibited 1.5 fold higher phytase activity (P < 0.0001) than that of hemizygous littermates. Moreover, no differential salivary phytase activity was seen in hemizygotes arising from CA-Yorkshire and CA-Duroc breed outcrosses, suggesting that expression of the transgene is unaffected by genetic background. This data demonstrates that an exogenous phytase gene can be stably transmitted and expressed in the salivary glands of a domestic food animal.

Application of iTRAQ to catalogue the skeletal muscle proteome in pigs and assessment of effects of gender and diet dephytinization.

In this study iTRAQ was used to produce a highly confident catalogue of 542 proteins identified in porcine muscle (false positive<5%). To our knowledge this is the largest reported set of skeletal muscle proteins in livestock. Comparison with human muscle proteome demonstrated a low level of false positives with 83% of the proteins common to both proteomes. In addition, for the first time we assess variations in the muscle proteome caused by sexually dimorphic gene expression and diet dephytinization. Preliminary analysis identified 19 skeletal muscle proteins differentially expressed between male and female pigs (> or = 1.2-fold, p<0.05), but only one of them, GDP-dissociation inhibitor 1, was significant (p<0.05) after false discovery rate correction. Diet dephytinization affected expression of 20 proteins (p<0.05). This study would contribute to an evaluation of the suitability of the pig as a model to study human gender-related differences in gene expression. Transgenic pigs used in this study might also serve as a useful model to understand changes in human physiology resulting from diet dephytinization.

Transgene and mitochondrial DNA are indicators of efficient composting of transgenic pig carcasses.

Composting is an environmentally sound method for the disposal of on-farm livestock mortalities that generates material suitable for use as fertilizer; however, this method is not generally permitted for disposal of transgenic livestock mortalities during the research and development phase. This study has explored the application of the polymerase chain reaction (PCR) as a method for assessing the persistence of transgene and mitochondrial DNA markers during the composting of euthanized transgenic pig. There was at least a 10(7) fold reduction of genetic material to a level that not either transgene or mitochondrion markers were detectable. At the end of the composting period, only bone fragments that were completely demineralised and chalky were detected. Chemically the compost was similar to that from pig litter and poultry mortalities, except the copper content was lower. Based on these data, composting appears to be an appropriate method for the disposal of transgenic animals.