RESUMO
Expression of foreign proteins in mammalian milk is becoming a widespread strategy for high-level production of recombinant pharmaceuticals, especially those with the most complex post-translational modifications. We have attempted in this project to develop transgenic mice harboring a transgene driving mammary gland expression of hybrid human salmon calcitonin. A milk-specific ovine beta-lactoglobulin [oBLG] promoter was used to drive expression of recombinant calcitonin in mouse milk. A gene construct was generated, consisting of 10.7kbp of the oBLG gene including its promoter and 3 flanking region with the calcitonin coding sequences inserted in-frame into the oBLG fifth exon. The gene construct was purified using CsCl gradient, released from vector, and gel-purified. After appropriate dilution, it was microinjected into recently-fertilized mouse oocytes. These oocytes then were transferred to pseudo-pregnant foster mice. Forty one pups were born from foster mice, which were genotyped using PCR, slot blotting, and Southern blotting. Among 9 mice which showed positive PCR results, 6 mice resulted in pups with positive PCR tests. All six families transmitted the transgene to first and second generation. As the main criteria for considering a mouse as transgenic is transgene transmission to the next generation, all 6 mice which stably transmitted their transgene to progeny are considered as transgenic founders and constitute independent transgenic lines