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

Abstract

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.

Cytosolic Delivery of Nanolabels Prevents Their Asymmetric Inheritance and Enables Extended Quantitative in Vivo Cell Imaging

“…2a, b) is solely due to dilution of the contrast agent upon subsequent cell divisions. Indeed, with a mono-exponential fit we obtained a cell division time of ~25 h for HeLa cells, which is in perfect agreement with reported values 47 .…”

Targeting Protein for Xenopus Kinesin-like Protein 2 (TPX2) Regulates γ-Histone 2AX (γ-H2AX) Levels upon Ionizing Radiation

“…Expression of TPX2-targeting miRNA in HeLa EM2-11-TPX2 was induced by doxycycline (1 g/ml of medium). Of note, although the doxycyclineinducible TPX2 RNAi originates from an exogenous miRNA, the mechanism of TPX2 knockdown is siRNA-based (17). Elements of miRNA precursors such as RNA polymerase II promotor, drosha processing, and Exportin 5 recognition sequences are used in this system to produce an siRNA double strand that is identical (apart from stabilizing modifications added to synthetic stealth siRNAs) to a synthetic siRNA duplex administered by transfection.…”

“…Moreover, TPX2 levels are altered in cancers associated with aberrant cellular responses to DNA damage and genomic instability (12)(13)(14)(15)(16)(17)(18)(19). These observations raise the question as to whether TPX2 is involved in the DNA damage response.…”

“…), and amplification of the TPX2 gene has been suggested to promote the progression of colorectal malignancies (12)(13)(14)(15)(16)(17)(18)(19). Conversely, TPX2 haploinsufficiency, leading to decreased levels of TPX2, significantly increases the propensity for the development of tumors in mice (20).…”

Streamlined platform for short hairpin RNA interference and transgenesis in cultured mammalian cells

“…This method eliminates the genome position effect and allows uniform transgene expression in independent recombinant clones. RMCE has recently been used for stable shRNA expression in HeLa cells and mouse embryonic stem cells (17)(18)(19)(20). However, the published techniques still rely on cell cloning to isolate shRNA-expressing cells from the original recombinant pools because of a background of unspecific integration events.…”