Human proinsulin production in the milk of transgenic cattle.

BACKGROUND: The worldwide growing demand for human insulin for treating diabetes could be supplied by transgenic animals producing insulin in their milk. METHODS AND RESULTS: Pseudo-lentivirus containing the bovine ß-casein promoter and human insulin sequences was used to produce modified adult fibroblasts, and the cells were used for nuclear transfer. Transgenic embryos were transferred to recipient cows, and one pregnancy was produced. Recombinant protein in milk was evaluated using western blotting and mass spectrometry. One transgenic cow was generated, and in milk analysis, two bands were observed in western blotting with a molecular mass corresponding to the proinsulin and insulin. The mass spectrometry analysis showed the presence of human insulin more than proinsulin in the milk, and it identified proteases in the transgenic milk that could convert proinsulin into insulin and insulin-degrading enzyme that could degrade the recombinant protein. CONCLUSION: The methodologies used for generating the transgenic cow allowed the detection of the production of recombinant protein in the milk at low relative expression compared to milk proteins, using mass spectrometry, which was efficient for detecting recombinant protein with low expression in milk. Milk proteases could act on protein processing converting recombinant protein to functional protein. On the other hand, some milk proteases could act in degrading the recombinant protein.

Effect of temporary meiosis block during prematuration of bovine cumulus-oocyte complexes on pregnancy rates in a commercial setting for in vitro embryo production.

Ovum pick up (OPU) associated with in vitro production (IVP) of embryos has been shown as an important tool in cattle breeding to increase the number of descendants from animals of high genetic value. In herds maintained distant from the laboratory, collecting cumulus-oocyte complexes (COCs) and transporting them to the laboratory may take several hours and decrease COCs viability, representing a challenge for commercial settings. In this study, a prematuration culture to induce temporary meiosis block was evaluated in a commercial scale IVP setting as a strategy to transport bovine OPU-derived COCs from Nelore and Brangus donors. Effects on embryo yield and pregnancy rates were assessed. Viable COCs from each donor were destined to one of the experimental groups (control, blocks 1 and 2). Control group COCs were placed in cryotubes with 1 mL TCM199-HEPES. In block groups (1 and 2), COCs were placed in cryotubes with 300 µL TCM 199 + 12 µM butyrolactone I (block medium). All groups were gassed and kept in a thermos bottle for 4 hours at 36 °C. Next, COCs in the control group were transferred to IVM medium and block 1 group to block medium, and cultured for 22 hours and 15 hours, respectively, at 38.5 °C and 5% CO2 in air. Block 2 COCs were kept in the cryotubes and in the thermos bottle for another 15 hours at 36 °C to simulate long-term transport conditions. After meiosis block in prematuration culture, blocks 1 and 2 COCs were matured in vitro for 22 hours as for the control group. After IVM, COCs in all groups were submitted to IVF and IVC, and blastocyst rates were evaluated on day 7. Embryos were transferred and pregnancy rates evaluated at 60 days of gestation. The mean total number of COCs retrieved by OPU did not differ between Nelore and Brangus donors (16.8 and 17.2, respectively, P > 0.05), but Nelore donors produced more viable COCs than Brangus (10.1 and 7.6, respectively, P < 0.05) and more embryos/cow (3.8 and 2.7, respectively, P < 0.05). Blastocyst rates were similar for control (40.2% and 36.7%), block 1 (37.3% and 34.5%), and block 2 groups (34.7% and 33.6%) for Nelore and Brangus cattle, respectively (P > 0.05). Pregnancy rates did not differ regardless of breed or treatment (36.7%, P > 0.05). In conclusion, temporary meiosis block during prematuration culture did not affect embryo development or pregnancy rates; therefore, this strategy may be used to transport bovine COCs in a commercial IVP setting.