Abstract
We have targeted the expression of recombinant human extracellular superoxide dismutase, a glycosylated tetrameric metalloprotein, to the mammary gland of transgenic mice. This was achieved by using regulatory elements from either the murine whey acidic protein gene or the ovine beta-lactoglobulin gene to control expression of human extracellular superoxide dismutase cDNA. Whey acidic protein regulatory sequences directed high level mammary gland-specific expression of the recombinant gene and secretion of biologically active extracellular superoxide dismutase into the milk. The produced recombinant protein was fully active, it was in tetrameric form, it showed heparin affinity, and its mass was similar to that of the native enzyme. In addition, the in vivo plasma clearance in a rabbit model was similar to the previously studied native and recombinant forms. To our knowledge, this is the first example of efficient production of a tetrameric, protease-susceptible metalloprotein in milk of transgenic animals. Production at equivalent levels in transgenic farm animals would yield sufficient extracellular superoxide dismutase for therapeutic purposes.
Highlights
9204 from the Swedish Natural Science Research Council
Virtually all EC-SOD exists anchored to heparan sulfate in the interstitial space and on the cell surfaces [8].The positively charged carboxyl-terminal end of the enzyme is responsible for the heparin binding [19]
The temporal and spatial distributionof recombinant gene expression directed by milk protein gene regulatory fragments from various species seems to be very conserved and general among all species studied [37,38].Despite the fact that many cDNAs and genomic fragments have been evaluated in mammary gland expression systems in transgenic animals, it is hard to define the factors that affect the level of expression
Summary
Subsequently hybridized to a 32P-labeledprobe according to the sup- Generationof TransgenicAnimals-To direct expression of plier’s protocol (Amersham, Little Chalfont, UnitedKingdom). A hybrid WAP/EC-SOD gene, pS172, was constructed. This recombinant gene comprises about 2.3 kb of upstream regulatory sequences including the first 24 bp of the transcribed but untranslated WAP sequence in front of the proteasespresentin the milk or released from cells disrupted by human EC-SOD cDNA. To generate WAP/EC-SOD transgenic animals, 178 injected ova at one-cell stage were implanted into 7 foster mothers resulting in 19 newborn mice. Expression of Recombinant EC-SOD mRNA-Expression of the transgenes was assessed by analyzing RNA and milk from lactating females that were either fo animals or the transgenic fi offsprings of fo males. The RNA hybridization results show expression in the lactating mammary gland of a 1-kb major EC-SOD mRNA species (Fig. 2a). The tissue distribution of recombinant WAP/EC-SOD expression was investigated in duplicate mice of the transgenic lines. Analysis of RNA prepared from various tissues (Fig. 2a) showed that abundant expression of recombinant EC-SOD was restricted to the lactating mammary gland, low levels of recombinant EC-SOD expression were a Mg U Ki Sp He Lu Sg Br
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