The eukaryotic genome contains chromosomal loci with a high transcription-promoting potential. For their identification in cultured cells, transfer of a reporter gene has to be performed by a technique that grants the integration of individual copies. We have applied retroviral vectors in conjunction with inverse polymerase chain reaction techniques to reconstruct a number of these sites for a further characterization. Remarkably, all examples conform to the same design in that the process of retroviral infection selected a scaffold- or matrix-attached region (S/MAR) that was flanked by DNA with high bending potential. The S/MARs are of an unusual type in that they show a high incidence of certain dinucleotide repeats and the potential to act as topological sinks. The anatomy of retroviral integration sites reveals principles that can be exploited for the development of predictable transgenic systems on the basis of expression and targeting vectors.
Most cell lines that are used for the production of recombinant proteins proliferate spontaneously at a high rate. In many types of cultivation systems these cells still keep growing after having reached the desired cell density. Further proliferation in batch cultures leads to cell death as a consequence of nutrient and oxygen depletion as well as to accumulation of lactate and toxic products. Consequently, in many technical processes, the surplus of cells is removed.We have established cell lines in which proliferation is controlled by a physiological regulator, IRF-1. IRF-1 (Interferon Regulatory Factor 1) is a transcriptional activator and acts as a tumor suppressor. Constitutive overexpression of recombinant IRF-1 leads to inhibition of cell growth. The extent of this growth arrest depends on the intracellular concentration of active IRF-1. To allow IRF-1 expression in various mammalian cells a system for conditional IRF-1 activation has been established. A fusion protein composed of IRF-1 and the hormone binding domain of the human estrogen receptor, was used. This system allows to control gradually the growth of several mammalian cell lines by adjusting the intracellular concentration of active IRF-1 via estradiol in the medium. We have evaluated BHK-21 cells with respect to IRF-1 mediated cell growth inhibition and expression of two secreted proteins. Whereas the productivity of proliferation inhibited cells with respect to constitutively transcribed IgG genes is reduced, productivity of another secreted protein which is controlled by an IRF-1 inducible promoter is strongly enhanced under these conditions.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.