Stable Integration and Functional Expression of Flounder Growth Hormone Gene in Transformed Microalga, Chlorella ellipsoidea

Abstract

Chlorella is an attractive organism for complex recombinant protein production because of its eukaryotic characteristics and low cost for large-scale culture. Protoplasts of C. ellipsoidea were transformed with a vector containing the flounder growth hormone gene (fGH) under the control of the cauliflower mosaic virus 35S promoter, and the phleomycin resistance Sh ble gene under the control of the Chlamydomonas RBCS2 gene promoter. The presence of introduced DNA was first determined by PCR amplification of both the fGH and Sh ble genes from genomic DNA isolated from transformants and fGH protein expression was detected by immunoblot analysis. Over 400 microg of fGH protein expression per one liter culture containing 1 x 10(8) cells/ml was estimated by ELISA. Stable integration of introduced DNA was confirmed by Southern blot analysis of genomic DNA digested with restriction enzymes. The introduced DNA and fGH expression were detected after seven successive transfers in media devoid of phleomycin, but stably remained in the presence of the antibiotic. Flounder fry fed on the transformed Chlorella revealed a 25% growth increase after 30 days of feeding.

Matrilin-1 Is an Inhibitor of Neovascularization

“…Cartilage is an avascular and relatively tumor-resistant tissue that is composed predominantly of proteoglycans, several types of collagen, and noncollagenous matrix proteins (1)(2)(3). The resistance of cartilage to capillary invasion has long been postulated to be a function of the presence of endogenous inhibitors of angiogenesis, which inhibit new blood vessel formation from pre-existing vessels.…”

“…The resistance of cartilage to capillary invasion has long been postulated to be a function of the presence of endogenous inhibitors of angiogenesis, which inhibit new blood vessel formation from pre-existing vessels. The first of these inhibitors purified to homogeneity was a cartilage-derived tissue inhibitor of metalloproteinases (TIMP), 2 a protein that was demonstrated to be a potent inhibitor of capillary EC proliferation and migration in vitro, and angiogenesis in vivo in both the chick chorioallantoic membrane (CAM) assay and the corneal pocket assay (4,5). Shortly thereafter, a chondrocyte-derived angiogenesis inhibitor, a 35.5-kDa protein isolated from chondrocyte primary cultures, was shown to inhibit angiogenesis in vitro in EC proliferation and migration assays and in vivo in the CAM assay (6).…”

A new inducible expression system in a transformed green alga, Chlorella vulgaris

“…These results demonstrated that CAT was successfully expressed in the transformed Chlorella and functioned properly to exhibit chloramphenicol resistance. An overall efficiency for our transformation was estimated to be approximately one transgenic colony per 3000 Chlorella cells, relatively high compared with any C. vulgaris nuclear transformation system ever reported (Apt et al, 1996;Chen et al, 2001;Kim et al, 2002). …”

“…In another study, C. ellipsoidea was transformed with a vector containing the flounder growth hormone gene (fGH) under the control of the 35S promoter, and the phleomycin resistance Sh ble gene under the control of the Chlamydomonas RBCS2 gene promoter. Over 400 µg fGH protein expression per one liter of culture containing 1 x 10 8 cells/mL was estimated by ELISA (Kim et al, 2002). However, the expression levels of these foreign genes in transformed Chlorella were still relatively low.…”

Les microalgues

“…Des centaines de milliers d'espèces de microalgues sont réparties sur la surface du globe en milieu marin, dulçaquicole 4 ou saumâtre (Figures 1-4 Dans la liste des promoteurs utilisés chez les microalgues, on trouve le très classique promoteur du gène 35S du virus de la mosaïque du chou-fleur, utilisé avec succès chez Chlorella ellipsoidea [19]. A mentionner, le promoteur de nitrate reductase endogène, actif chez Chlamydomonas processus de glycosylation, en amont du point de divergence entre les plantes supérieures et les animaux.…”