Introduction: 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 calcionin. Materials and Methods: 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. Results: 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. Conclusion: 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.    

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. A gene construct was generated, consisting of 10.7 kbp of the ovine beta-lactoglobulin (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. It was microinjected into fertilized mouse oocytes. These oocytes were then transferred to pseudo-pregnant foster mice. The pups born from foster mice were genotyped using PCR, slot blot, and Southern blot techniques. Among 9 mice which showed positive PCR results, only 6 mice transmitted the Transgene to the next generation. Therefore, 6 transgenic lines were established which stably transmitted their Transgene to their progeny.

To investigate the impact of overexpression ofAtNRT2.1 Transgene from Arabidopsis on nitrate uptake rate and to understand the regulation of endogenous HATS by nitrate and glutamine amino acid (Gln) in tobacco plants, wild-type and transgenic (F line) plants grown on soil for 4 weeks were transferred to hydroponic culture in a controlled-environment with a 16/8h L: D photoperiod at 24oC/20oC, 70% relative humidity and 150 mmol. m-2. s-1 light intensity. Nitrate uptake over time was studied by the ion depletion method. Nitrate uptake in the 2 h exposure to 150 µM nitrate showed an increase in the transgenic line compared to the wild-type plant. Subsequently the uptake trend followed a similar pattern in wild-type plant and transgenic plants. Pretreatment with high concentrations of nitrate (10 mM) increased rate of uptake in both wild-type and transgenic plants.No marked differences between the two genotypes were observed with respect to HATS activity.Glutamine decreased the rate of nitrate uptake in both wild-type and, to a lesser extent, in transgenic plants.