Secretion of recombinant human fibrinogen by the murine mammary gland.

Transgenic animals secreting individual chains and assembled fibrinogen were produced to evaluate the capacity of the mammary gland for maximizing assembly, glycosylation and secretion of recombinant human fibrinogen (rhfib). Transgenes were constructed from the 4.1 kbp murine Whey Acidic Protein promoter (mWAP) and the three cDNAs coding for the Aalpha, Bbeta and gamma fibrinogen chains. Transgenic mice secreted fully assembled fibrinogen into milk at concentrations between 10 and 200 microg/ml, with total secretion of subunits approaching 700 microg/ml in milk. Partially purified fibrinogen was shown to form a visible and stable clot after treatment with human thrombin and factor XIII. The level of assembled fibrinogen was proportional to the lowest amount of subunit produced where both the Bbeta and gamma chains were rate limiting. Both the Bbeta and gamma chains were glycosylated when co-expressed and the degree of saccharide maturation was dependent on expression level, with processing preferred for gamma chains over Bbeta chains. Also, the subunit complexes gamma2, Aalphagamma2 and the individual subunits Aalpha, Bbeta and gamma were found as secretion products. When the Bbeta was secreted individually, the glycosylation profile of the molecule was of a mature complex saccharide indicating recognition of the molecule by the glycosylation pathway without association with other fibrinogen chains. To date secretion of Bbeta chain has been not observed in any cell type, suggesting that the secretion pathway in mammary epithelia is less restrictive than that occurring in hepatocytes and other cells previously used to study fibrinogen assembly.

Transgenic mice expressing recombinant human protein C exhibit defects in lactation and impaired mammary gland development.

To determine if the production of recombinant human protein C (rHPC) could be increased in milk, we created two lines of mice homozygous for the mouse whey acidic protein (WAP)/human protein C (HPC) transgene. Females of both lines had normal growth, activity and fertility, but failed to lactate normally and were unable to raise litters. Histological analyses of mammary glands from lactating homozygous females showed barely distended alveoli filled with dense-staining milk. Epithelial cells within these alveoli had distinct, centrally located nuclei and contained intracellular lipid droplets. Hemizygous animals derived from these lines were able to lactate and raised normal sized litters. Northern blot analysis showed that the 6.4 homozygous (6.4H) line expressed the transgene at higher levels then corresponding hemizygous (6.4) animals, but the 4.2 homozygous (4.2H) line expressed the transgene at lower levels than the 4.2 hemizygous line. The 6.4H line also had increased rHPC levels in the milk as revealed by western blot analysis. The 4.2H, 6.4, and 6.4H lines showed decreased and/or delayed expression of WAP, beta-casein, and alpha-lactalbumin mRNA's compared to wild type animals during lactogenesis. The 4.2 line showed decreased mRNA expression for beta-casein and alpha-lactalbumin, but normal or higher expression of WAP during lactogenesis. Elevated levels of some proteins were detected in the milk of transgenic mice. From these results, it is concluded that expression of rHPC induced a lactational phenotype that involves abnormal morphological, biochemical, and functional differentiation of mammary epithelial cells. However, the induction of this phenotype does not appear to be directly related to the level of rHPC mRNA expression, thus suggesting that the basis of this phenotype may involve secondary, rather than primary, effects of rHPC on mammary gland development.

The use of the uromodulin promoter to target production of recombinant proteins into urine of transgenic animals.

A uromodulin promoter has been isolated, sequenced, and used to generate two sets of transgenic mice for expression of the lacZ marker gene and for production of the human recombinant erythropoietin (rhEPO) in urine. We demonstrated that the 5.6-kb fragment of the uromodulin gene containing the 3.7-kb promoter area and, both the first exon and part of the second exon, were sufficient to provide kidney-specific expression of the lacZ gene. Histological analysis of the lacZ expression pattern revealed beta-galactosidase activity specifically in the thick limb of Henle's loop. However, due to random integration of the transgene, ectopic expression was detected in some transgenic lines. Analysis of the EPO-transgenic mice showed that rhEPO was secreted into the urine of founder mice (up to 6 ng/ml). We were able to breed and analyze only two sublines with a very low expression level of rhEPO (up to 260 pg/ml). All of our transgenic mice expressing rhEPO in urine developed disease symptoms similar to polycythemia in humans. These included a considerable increase in red blood cell counts, hemoglobin concentration, and hematocrit concomitant with severe thrombocytopenia, all of which were detected in the rhEPO-expressing mice. Although our model did not prove to be beneficial for commercial production of rhEPO, we concluded that the uromodulin promoter could be useful for expression of other important therapeutic proteins into the urine of transgenic animals.

Uromodulin promoter directs high-level expression of biologically active human alpha1-antitrypsin into mouse urine.

We have recently shown that the regulatory sequence of the uromodulin gene, containing the 3.7 kb promoter, exon 1 and a part of exon 2, provided for kidney-specific expression of the reporter lacZ gene in transgenic mice [Zbikowska, Soukhareva, Behnam, Chang, Drews, Lubon, Hammond and Soukharev (2002) Transgenic Res., in the press]. In the present study, we generated transgenic mice harbouring the regulatory sequence of the uromodulin gene to direct the expression of human alpha1-antitrypsin (alpha1AT) into urine. Of the 13 founder mice that tested positive by PCR, seven showed the presence of the human protein in their urine. The concentration of the recombinant human (rh) alpha1AT in the urine, estimated by using ELISA, ranged from 0.5 to 14 microg/ml in the F(0)-generation mice, and reached up to 65 microg/ml in the F1 generation. The transgenically produced rh alpha1AT was found to be N-glycosylated and biologically active. The N-terminal sequence analysis confirmed the identity of the human protein and revealed that the recombinant alpha1AT was correctly processed with the signal peptide cleaved off. Our results demonstrate for the first time that the uromodulin regulatory sequence provides a very attractive option for the potential large-scale production of functional therapeutic proteins in livestock.

Changes in liver nuclear sap proteins during chick embryo development.

Nuclear sap proteins from liver of 12-, 15-, 19-day-old embryos and 1-day-old chicks were resolved by one-and two-dimensional gel electrophoresis. Although the protein patterns from various stages of development have remarkable similarities, some qualitative and quantitative differences were found among these patterns. The most pronounced changes were detected in protein with molecular weight of 100 K which was very abundant in nuclei of 12-day-old embryos and disappeared in nuclei of older embryos and in protein with molecular weight of 40 K which rapidly diminished after hatching.