La ß-Lactoglobulina y su aplicación en transgénesis

Resumo

The final aim of this project is to produce pharmaceutical recombinant proteins in milk of farm animals. Previous works of our group have been performed to study the caprine b-lactoglobulin (bLG) regulatory sequences resulting in a expression cassette that is able to target the expression of the transgene to the mammary gland of transgenic mice in an integration-site dependent manner, independently of the number of copies in the array. All the constructions have been previously tested in a mouse model due to their short generation intervals and ease of genetic manipulation being cheaper than farm animals. In the present work, a goat bLG expression cassette which contained 410 bp of proximal promoter region and 2.5 kb of 3 flanking region has been used to target the expression of the two subunits (a and b) of human Follicle Stimulating Hormone (hFSH) to the mammary gland of transgenic mice. Four established transgenic lines that express both hFSH transgenes (a and b) to their mammary gland have been obtained. On the other hand, homozygous mice from one (tg56) of the different transgenic lines, that have been previously obtained for the goat bLG gene to study the regulatory sequences, displayed a distinct phenotype. Here, we present the identification and characterization of the transgene-induced insertional mutation at the PLC-b1 locus (PLC-b1bLG) in line tg56 of bLG transgenic mice. Furthermore, a rapid and accurate real-time quantitative PCR-based system to determine transgene copy number in transgenic animals has been developed. This method can be used directly for the analysis of transgenic mice for transgenes with different ruminant sequences without the requirement of control sample previously quantified by blotting techniques. Finally, fifteen polymorphisms, nine in the promoter region, of the caprine bLG gene have been identified and characterized. The main aim of this study has been identified mutations in the proximal promoter region that could be important in the expression of the gene.