WAP is being recognized as the principal milk protein expressed in pregnant or lactating females of several mammalian species. Recently, it has been shown that the 6.3-kb 5' untranslated region of the rWAP gene is able to control, and almost completely restrict, the expression of the transgene into the mammary gland of the transgenic animal. We cloned the genomic fragment carrying the rWAP gene locus from the rabbit phage genomic library and used the 8.5-kb long 5' untranslated part of the rWAP gene to target the expression of hEPO, cloned from the human phage genomic library, into the mammary gland of the mouse. The vectors, carrying either the hEPO gene or the rWAP-hEPO hybrid gene, were injected into the mouse ova, and 12 transgenic animals were identified by PCR and Southern blot from the progeny of 168 tested littermates. Transgenic mice were viable, fertile and displayed a normal development. Recombinant human erythropoietin was produced in the milk of a transgenic mouse female at a secretion level of 5.3 mIU/ml, as detected by ELISA. Despite the low production of the transgenic glycoprotein in the milk we demonstrate that the hybrid gene can be expressed in the mammary gland of the host animal. Thus, WAP-based recombinant vectors, with additional optimizing modifications, can be useful for production of therapeutic proteins in the transgenic mammals.

Biopharm Research Institute of Biopharmacy and Veterinary Drugs Inc Jíflové u Prahy Czech Republic

Generation and phenotypic analysis of a transgenic line of rabbits secreting active recombinant human erythropoietin in the milk