Toxin–antitoxin based transgene expression in mammalian cells

Long-term, recombinant gene expression in mammalian cells depends on the nature of the transgene integration site and its inherent properties to modulate transcription (epigenetic effects). Here we describe a method by which high transgene expression is achieved and stabilized in extensively proliferating cultures. The method is based on strict co-expression of the transgene with an antitoxin in cells that express the respective toxin. Since the strength of antitoxin expression correlates with an advantage for cell growth, the cells with strong antitoxin expression are enriched over time in cultures of heterogeneous cells. This principle was applied to CHO cell lines that conditionally express the toxin kid and that are transduced to co-express the antitoxin kis together with different transgenes of interest. Cultivation of pools of transfectants that express the toxin steadily increase their transgene expression within several weeks to reach a maximum that is up to 120-fold over the initial status. In contrast, average transgene expression drops in the absence of toxin expression. Together, we show that cells conditionally expressing kid can be employed to create overexpressing cells by a simple coupling of kis to the transgene of interest, without further manipulation and in absence of selectable drugs

Generation and characterization of two immortalized human osteoblastic cell lines useful for epigenetic studies

Different model systems using osteoblastic cell lines have been developed to help understand the process of bone formation. Here, we report the establishment of two human osteoblastic cell lines obtained from primary cultures upon transduction of immortalizing genes. The resulting cell lines had no major differences to their parental lines in their gene expression profiles. Similar to primary osteoblastic cells, osteocalcin transcription increased following 1,25-dihydroxyvitamin D3 treatment and the immortalized cells formed a mineralized matrix, as detected by Alizarin Red staining. Moreover, these human cell lines responded by upregulating ALPL gene expression after treatment with the demethylating agent 5-aza-2 Œ-deoxycytidine (AzadC), as shown before for primary osteoblasts. We further demonstrate that these cell lines can differentiate in vivo, using a hydroxyapatite/tricalcium phosphate composite as a scaffold, to produce bone matrix. More importantly, we show that these cells respond to demethylating treatment, as shown by the increase in SOST mRNA levels, the gene encoding sclerostin, upon treatment of the recipient mice with AzadC. This also confirms, in vivo, the role of DNA methylation in the regulation of SOST expression previously shown in vitro. Altogether our results show that these immortalized cell lines constitute a particularly useful model system to obtain further insight into bone homeostasis, and particularly into the epigenetic mechanisms regulating sclerostin production