Production and characterization of guinea pig IL-5 in baculovirus-infected insect cells.

To study the role interleukin (IL)-5 may play in altering airway function in asthma, we have produced recombinant protein for exogenous administration to guinea pigs. The guinea pig IL-5 (gpIL-5) cDNA was cloned by polymerase chain reaction (PCR) amplification of guinea pig spleen RNA and expressed as a secretion product from recombinant baculovirus-infected Sf9 insect cell cultures. The protein was purified to homogeneity by a four-step procedure that included immunoaffinity chromatography using polyclonal antipeptide antibodies against a region of the mature secreted cytokine. The cytokine was properly processed after the signal sequence by the Sf9 cells, was glycosylated with terminal mannose-containing oligosaccharide, and had proper disulfide-linked dimer structure as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified preparation was active in vitro and in vivo as determined by its ability to prime human basophils to release leukotriene C4 in the presence of C5a and to induce airway eosinophilia in naive guinea pigs.

Expression of chloramphenicol acetyltransferase (CAT) from rodent type I collagen promoters in transfected osteosarcoma cells in vivo.

We examined the osteoblastic phenotype of permanently transfected ROS 17/2.8 cells in culture and in vivo, in order to evaluate their relevance for studies of the regulation of gene expression and gene function in osteoblastic cells. Recent reports indicate that the progeny transfected cells may substantially vary and differ from the parental cell line in their phenotype, particularly in their tumorigenicity. ROS 17/2.8 cells were transfected with genetic constructs expressing the CAT gene from either the rat alpha 1 (I) or the mouse alpha 2 (I) collagen promoters. Forty-four clonal cell lines display a range of CAT expression from the transfected collagen promoters in culture. Four of these cell lines were further characterized. Alkaline phosphatase activity in these four cell lines is higher than in fibroblastic cells. These four cell lines are tumorigenic in immunocompatible ACI rats and form calcified tumors similar to those formed by ROS 17/2.8. CAT expression could be demonstrated in tumor extracts of two of the four cell lines, which also expressed higher CAT levels in culture. We conclude that permanently transfected ROS 17/2.8 derived cell lines maintain their tumorigenicity and their osteoblastic-like phenotype, and thus may provide a useful system for studies of gene function and regulation in osteoblast-like cells and bone-like tissue in vivo.

Isolation and characterization of the rat alpha 1(I) collagen promoter. Regulation by 1,25-dihydroxyvitamin D.

Our previous work demonstrated that the inhibition of type I collagen synthesis by 1,25-dihydroxyvitamin D (1,25-(OH)2D3) in fetal rat calvaria and cultured rat osteosarcoma cells is accompanied by equivalent reduction in steady state levels of alpha 1(I) and alpha 2(I) collagen mRNA. To pursue the mechanism for this effect, we isolated and sequenced a 3.6-kilobase DNA fragment that contained the promoter for the rat alpha 1(I) collagen gene. This promoter fragment was fused to the chloramphenicol acetyltransferase gene and was introduced into ROS 17/2.8 cells by calcium phosphate co-precipitation. Expression of this construct was diminished by 1,25-(OH)2D3 to the same degree as the endogenous collagen gene in both transient expression assays and in permanently selected bone cells. However, a fibroblast cell line did not show a similar reduction in the activity of the transgene or the endogenous collagen gene. These experiments indicate that the alpha 1(I) promoter contains cis-active elements which are regulated by the 1,25-(OH)2D3 receptor in ROS 17/2.8 cells.