Inducible expression of coding and inhibitory RNAs from retargetable genomic loci

Conditional gene expression systems have developed into essential tools for the study of gene functions. However, their utility is often limited by the difficulty of identifying clonal cell lines, in which transgene control can be realized to its full potential. Here, we describe HeLa cell lines, in which we have identified—by functional analysis—genomic loci, from which the expression of transgenes can be tightly controlled via tetracycline-regulated expression. These loci can be re-targeted by recombinase-mediated cassette exchange. Upon exchange of the gene of interest, the resulting cell line exhibits the qualitative and quantitative properties of controlled transgene expression characteristic for the parent cell line. Moreover, by using an appropriate promoter, these cell lines express the tetracycline controlled transcription activator rtTA2-M2 uniformly throughout the entire cell population. The potential of this approach for functional genomics is highlighted by utilizing one of our master cell lines for the efficient microRNA-mediated knockdown of the endogenous human lamin A/C gene

Quantitative analysis of conditional gene inactivation using rationally designed, tetracycline-controlled miRNAs

The combination of RNA interference (RNAi) with the tetracycline-controlled transcription activation (tet) system promises to become a powerful method for conditional gene inactivation in cultured cells and in whole organisms. Here, we tested critical sequence elements that originated from miRNA mR-30 for optimal efficiency of RNAi-based gene knockdown in mammalian cells. Rationally designed miRNAs, expressed conditionally via the tet system, led to an efficient knockdown of the expression of both reporter genes and the endogenous mitotic spindle protein TPX2 in HeLa cells. Quantitative studies of the tet-controlled gene inactivation revealed that the residual expression of the target gene is an intrinsic attribute of all cells that cannot be eliminated either by increasing the miRNA to target mRNA ratio or by simultaneous expression of miRNAs targeting different sequences within the transcript. The kinetic analysis of the reversibility of the miRNA mediated knockdown suggests that the recovery of target gene expression is primarily driven by cell division. Our miRNA design provides a useful tool for conditional gene inactivation in combination with the RNA-polymerase II based tet system. The identified characteristics of the conditional RNAi-mediated knockdown need to be considered for its application in cell culture or in vivo

Deep tissue volumetric optoacoustic tracking of individual circulating tumor cells in an intracardially perfused mouse model

Widespread metastasis is the major cause of death from melanoma and other types of cancer. At present, the dynamic aspects of the metastatic cascade remain enigmatic. The feasibility to track circulating melanoma cells deep within living intact organisms can greatly impact our knowledge on tumor metastasis, but existing imaging approaches lack the sensitivity, spatio-temporal resolution or penetration depth to capture flowing tumor cells over large fields of view within optically-opaque biological tissues. Vast progress with the development of optoacoustic tomography technologies has recently enabled two- and three-dimensional imaging at unprecedented frame rates in the order of hundreds of Hertz, effectively mapping up to a million image voxels within a single volumetric snapshot. Herein, we employ volumetric optoacoustic tomography for real-time visualization of passage and trapping of individual B16 melanoma cells in the whole mouse brain. Detection of individual circulating melanoma cells was facilitated by substituting blood with an artificial cerebrospinal fluid that removes the strong absorption background in the optoacoustic images. The approach can provide new opportunities for studying trafficking and accumulation of metastatic melanoma cells in different organs.ISSN:1522-8002ISSN:1476-558