A convenient method for the identification and expression of eukaryotic genes

The sequences of the cDNA variant lumbrokinase and the genomic DNA of beta-casein gene expression regulatory elements have been submitted to the GenBank(R), DDBJ, EMBL and GSDB Nucleotide Sequence Databases under the accession numbers AY327442C and AY311384 respectively. In order to identify whether the product of a genetically modified or newly isolated eukaryotic gene has biological activity, the gene of interest is usually subcloned into a mammalian expression vector and then expressed in an in vitro system such as in tissue culture. In the present study an efficient in vivo system has been developed by employing a mammary-gland-specific vector and expressing the targeted protein in the lactating-goat mammary glands. In this system, the synthesized lumbrokinase cDNA variant (LK-m) and the tissue-type plasminogen activator (tPA) cDNA were selected as genes of interest and cloned downstream of the goat beta-casein regulatory sequence. The LK-m- and tPA-expressing plasmids were prepared to high purity and portions (100-800 microg) injected into lactating-goat mammary-gland tissues. High-level expression of the LK-m and tPA was detected, by a fibrin-agarose plate assay, as fibrin-lysis activity. A dynamic study showed that the specific expression starts immediately after injection, generally reaches peak in 6-9 h, persists for 20-24 h at peak and the expression lasts for 4 days with gradual decline in the amounts expressed. The potential use of this system as bioreactor for the production of biological proteins in place of transgenic animals is implicated from this study.