M.J. Sleigh

A nonchromatographic assay for expression of the chloramphenicol acetyltransferase gene in eucaryotic cells

A rapid procedure is described for assaying chloramphenicol acetyltransferase (CAT) enzyme activity following transfection of the CAT gene into eucaryotic cells. CAT enzyme activity in cell extracts catalyzes the transfer of [14C]acetyl groups from labeled acetyl coenzyme A to unlabeled chloramphenicol. Labeled reaction product is quantitated by liquid scintillation counting after extraction into ethyl acetate. The method is valid for use with transfected cell extracts only if the extracts are first heated to 65 degrees C to remove a factor which degrades acetyl coenzyme A. The revised procedure offers considerable advantages in speed and ease of performance over the chromatographic assay in current use.

Super-CHO - A cell line capable of autocrine growth under fully defined protein-free conditions

Chinese Hamster Ovary (CHO) cells are widely used for the large scale production of recombinant biopharmaceuticals. Growth of the CHO-K1 cell line has been demonstrated in serum-free medium containing insulin, transferrin and selenium. In an attempt to get autocrine growth in protein-free medium, DNA coding for insulin and transferrin production was transfected into CHO-K1 cells. Transferrin was expressed well, with clones secreting approximately 1000 ng/106 cells/24h. Insulin was poorly expressed, with rates peaking at 5 ng/106 cells/24h. Characterisation of the secreted insulin indicated that the CHO cells were incompletely processing the insulin molecule. Site-directed mutagenesis was used to introduce a furin (prohormone converting enzyme) recognition sequence into the insulin molecule, allowing the production of active insulin. However, the levels were still too low to support autocrine growth. Further investigations revealed insulin degrading activity (presumably due to the presence of insulin degrading enzymes) in the cytoplasm of CHO cells. To overcome these problems insulin-like growth factor I (instead of insulin) was transfected into the cells. IGF-1 was completely processed and expressed at rates greater than 500 ng/106cells/24h. In this paper we report autonomous growth of the transfected CHO-K1 cell line expressing transferrin and IGF-1 in protein-free medium without the addition of exogenous growth factors. Growth rates and final cell densities of these cells were identical to that of the parent cell line CHO-K1 growing in insulin, transferrin, and selenium supplemented serum-free media.

TATA box‐independent transcription of the human tissue plasminogen activator gene initiates within a sequence conserved in related genes

Transcription of the human tissue‐type plasminogen activator (tPA) gene has been reported to initiate from a single site proximal to a TATA box motif (1985, J. Biol. Chem. 260, 11223–11230). In this study, we utilized primer extension analysis to evaluate the tPA mRNA start site in phorbol‐12‐myristate 13‐acetate (PMA) induced WI‐38 man lung fibroblast cells. Whilst some tPA mRNA initiated from the predicted TATA‐proximal location (+1), a 10‐fold greater proportion of tPA mRNA transcripts initiated 110 bases downstream from a sequence conserved and utilized as the TATA‐independent transcription start site in the rodent tPA genes. Moreover, the transfection and expression in different cell types of a cosmid containing the entire human tPA gene resulted in utilization of the same downstream (+110) start site. We propose that this, rather than the previously published position, is the major transcriptional initiation point for the human tPA gene. A core sequence (5′‐CAGAGCTG‐3′) was identified which is common to the TATA‐independent mRNA start sites of the human, mouse and rat tPA genes, and which demonstrates only partial similarity to sequences found at the initiation point of other TATA‐independent genes.

Hyperinducible gene expression from a metallothionein promoter containing additional metal-responsive elements

We describe the development of metallothionein-based vectors with low basal levels of expression that are hyperinducible upon treatment with heavy metals. Vectors were constructed by substituting a region in the hMTIIA promoter (bp -70 to -129) containing an element (BLE) involved in basal level expression with multiple metal responsive elements (MREs). In expression studies utilizing cat as a reporter gene, heavy metal inducibility was examined in both transiently transfected and permanently transformed Chinese hamster ovary (CHO) cells. Our results demonstrate that, within the same promoter structure, inducibility can be increased by altering the ratio of MREs to BLEs. Optimal induction of expression in permanently transformed CHO cells was achieved by exposure to heavy metals for 48 h prior to cell harvest, with an additional boost 12 h before harvest. These vectors have the potential to be used for production of proteins in cultured mammalian cells and in gene expression in transgenic animals.

Extracellular matrix is a source of mitogenically active platelet-derived growth factor

Platelet‐derived growth factor (PDGF) is a chemotactic and mitogenic agent for fibroblasts and smooth muscle cells and plays a key role in the development of atherosclerotic lesions. PDGF is produced by a number of normal and transformed cell types and occurs as homo‐ or heterodimers of A and B polypeptide chains. Using Chinese hamster ovary (CHO) cells transfected with various forms of PDGF, we have previously shown that PDGF As (short splice version) is secreted, PDGF AL (long splice version) predominantly extracellular matrix‐associated, and PDGF B divided between medium, cells, and matrix. In the present study we have demonstrated the mitogenic activity of matrix‐localized PDGF in artificial and more physiologically relevant models by culturing Balb/c‐3T3 cells (3T3), human foreskin fibroblasts (HFF), and rabbit aortic smooth muscle cells (SMC) on extracellular matrix (ECM) laid down by PDGF‐expressing CHO cells and human umbilical vein endothelial cells (HUVEC). These cells responded to the local growth stimulus of PDGF‐containing CHO ECM and HUVEC ECM. We showed that 3T3 cells required proteolytic activity to utilize matrix‐localized PDGF, as aprotinin and η‐ACA inhibited growth and 3T3 cells were shown to possess plasminogen activator activity. HFF and SMC did not appear to require proteolytic activity (including metalloproteinase and serine protease activity) as a prerequisite for mitogenesis but were able to access immobilized PDGF by contact with the matrix. An understanding of the mechanisms whereby the utilization of stored PDGF is controlled in situations of excessive cellular proliferation will aid in the development of therapy for these conditions.

Chinese hamster ovary cells produce sufficient recombinant insulin-like growth factor I to support growth in serum-free medium. Serum-free growth of IGF-I-producing CHO cells

Insulin-like growth factor I has similar mitogenic effects to insulin, a growth factor required by most cells in culture, and it can replace insulin in serum-free formulations for some cells. Chinese Hamster Ovary cells grow well in serum-free medium with insulin and transferrin as the only exogenous growth factors. An alternative approach to addition of exogenous growth factors to serum-free medium is transfection of host cells with growth factor-encoding genes, permitting autocrine growth. Taking this approach, we constructed an IGF-I heterologous gene driven by the cytomegalovirus promoter, introduced it into Chinese Hamster Ovary cells and examined the growth characteristics of Insulin-like growth factor I-expressing clonal cells in the absence of the exogenous factor. The transfected cells secreted up to 500 ng/106 cells/day of mature Insulin-like growth factor I into the conditioned medium and as a result they grew autonomously in serum-free medium containing transferrin as the only added growth factor. This growth-stimulating effect, observed under both small and large scale culture conditions, was maximal since no further improvement was observed in the presence of exogenous insulin.

Oncogene expression in differentiating F9 mouse embryonal carcinoma cells

Differentiation of F9 mouse embryonal carcinoma cells in culture is accompanied by a decrease in growth rate and loss of tumorigenicity. Cells differentiating in monolayer culture (to parietal endoderm-type cells) or in aggregates (to visceral endoderm-type cells) show qualitatively similar changes in transcript levels from several c-oncogenes. In contrast with other studies with F9 cells, we find an early decrease in c-myb RNA but not in c-myc RNA. This and a later increase in c-src RNA may be associated with decreasing cell growth rate. Before differentiation, induction and maintenance of elevated c-abl RNA levels depend on the presence of retinoic acid in the medium. After differentiation c-abl RNA levels decline only partially when retinoic acid is removed. Increased RNA from c-fos is seen late in differentiation in monolayer cultures only, a change also seen with appearance of similar endoderm cell types in the developing mouse embryo.

Virus expression as a probe of regulatory events in early mouse embryogenesis

Abstract Many viruses exist in a silent form in cells of the early mouse embryo. Activation of viral gene expression in associated with the appearance of particular cell types during development. Thus viruses can be used to probe mechanisms regulating switches in gene expression during some of the steps of embryogenesis.

Processing of mutated human proinsulin to mature insulin in the non-endocrine cell line, CHO

Heterologous genes encoding proproteins, including proinsulin, generally produce mature protein when expressed in endocrine cells while unprocessed or partially processed protein is produced in non-endocrine cells. Proproteins, which are normally processed in the regulated pathway restricted to endocrine cells, do not always contain the recognition sequence for cleavage by furin, the endoprotease specific to the constitutive pathway, the principal protein processing pathway in non-endocrine cells. Human proinsulin consists of B-Chain — C-peptide — A-Chain and cleavage at the B/C and C/A junctions is required for processing. The B/C, but not the C/A junction, is recognised and cleaved in the constitutive pathway. We expressed a human proinsulin and a mutated proinsulin gene with an engineered furin recognition sequence at the C/A junction and compared the processing efficiency of the mutant and native proinsulin in Chinese Hamster Ovary cells. The processing efficiency of the mutant proinsulin was 56% relative to 0.7% for native proinsulin. However, despite similar levels of mRNA being expressed in both cell lines, the absolute levels of immunoreactive insulin, normalized against mRNA levels, were 18-fold lower in the mutant proinsulin-expressing cells. As a result, there was only a marginal increase in absolute levels of insulin produced by these cells. This unexpected finding may result from preferential degradation of insulin in non-endocrine cells which lack the protection offered by the secretory granules found in endocrine cells.