Transgenesis for the study and the control of lactation.

The study and the control of milk synthesis are required to decipher the mechanisms of gene expression, to improve milk production, to modify milk composition, to induce a resistance to diseases in the mammary gland and to produce recombinant proteins of pharmaceutical interest. Transgenesis has become a mandatory tool to reach these goals. The use of transgenesis is still limited by the difficulty of adding foreign genes in farm animals and mainly by replacing genes by homologous recombination. Transgene expression is also often ill-controlled. The present paper summarizes the current progress in this field with a particular emphasis on expression vectors for transgenes.

Position-independent and tissue-specific expression of porcine whey acidic protein gene from a bacterial artificial chromosome in transgenic mice.

Silencing of transgenes is a frequent event after the random integration of foreign DNA in the host genome following microinjection. Long genomic fragments are expected to contain all the regulatory elements necessary to induce an appropriate expression of transgenes. A bacterial artificial chromosome containing the porcine wap gene with approximately 145 and 5 kb of 5'- and 3'-flanking sequences, respectively, was microinjected into fertilized mouse ovocytes. In the six transgenic lines studied, expression was strictly specific to the mammary gland of lactating animals and was position-independent. Levels of exogenous porcine wap mRNA per copy compared favorably with the porcine wap mRNA yield in the mammary gland of a 9-day lactating pig. These findings suggest that this insert contained most if not all of the cis-acting elements involved in the full specific expression of the porcine wap gene. These elements constitute good candidates for directing the optimized expression of protein recombinant-encoding genes in the mammary gland of lactating animals.

The insulator effect of the 5'HS4 region from the beta-globin chicken locus on the rabbit WAP gene promoter activity in transgenic mice.

Previous studies have shown that the 5'HS4 DNaseI hypersensitive site of the chicken beta-globin locus is endowed with classic insulator activities: (i) it blocks the interaction between promoter and enhancers when it is inserted between them (ii) it confers expression of integrated foreign genes independent of their position in the chromatin. The aim of this present work was to determine whether the 5'HS4 element was able to stimulate the expression level and/or to increase the expression frequency of a luc+ reporter gene controlled by the rabbit WAP gene promoter. Two constructs with 5'HS4 insulator (p5'HS4-WAPluc) or without (pWAPluc) were introduced in mouse fertilised oocytes. All transgenic lines containing the 5'HS4 element (six lines) expressed the transgene whereas only two out of eight lines harbouring the pWAP-luc construct expressed the transgene to a significant level. Moreover, the mean level of expression was seven times higher in p5'HS4WAP-luc lines than in pWAP-luc lines. Even all these benefits on transgene expression, the 5'HS4 element did not confer a copy-dependent expression, did not decrease the ectopic expression of the reporter gene and did not decrease the variability of expression. Thus, the 5'HS4 element does not have all the properties of a perfect insulator on a construct containing the luc+ reporter gene controlled by the rabbit WAP promoter.