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A convenient method for the identification and expression of eukaryotic genes
Author(s) -
Hu Rongliang,
Zhang Shoufeng,
Xu Qing,
Tu Changchun
Publication year - 2004
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1042/ba20030153
Subject(s) - complementary dna , biology , microbiology and biotechnology , gene , gene expression , expression vector , genomic dna , biochemistry , recombinant dna
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 β‐casein regulatory sequence. The LK‐m‐ and tPA‐expressing plasmids were prepared to high purity and portions (100–800 μg) 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.