11.07.2008

A drug-inducible transgenic system for direct reprogramming of multiple somatic cell types

Wernig et al. Nat Biotechnol. 2008 Jul.1 Epub ahead of print

Comment:

Pluripotency can be induced in somatic cells by ectopic expression of transcription factors Oct4, Sox2, Klf4 and Myc. So far this is done by infecting fibroblasts with high-titer retroviral vectors, which results in reprogramming and leads to the formation of induced pluripotent stem cells (iPS) at a low frequency. In a recent publication members from R.Jaenisch’s lab at MIT have significantly enhanced this process by using genetically modified, homogenous fibroblasts and Tet- regulated expression of reprogramming factors.

20.03.2008

Site-directed, virus-free, and inducible RNAi in embryonic stem cells

Wang, J. et al. 2007 PNAS 104 (52): 20850-20855

Comment:

In a recent report published in PNAS, researchers from the Orkin laboratory at Harvard established an efficient system for Tetracyline- inducible RNAi in murine ES cells by combining the miRNA approach with a locus-defined expression strategy: the shRNA-mir expression cassettes under control of a tetracycline (tet)-regulatable promoter were targeted close to the constitutively active hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus through Cre mediated site-specific recombination. In their experimental setup, the Tet-On transactivator (rtTA) was driven by the endogenous Rosa26 locus, thus ensuring ubiquitous expression. A single microRNA embedded shRNA hairpin was sufficient to efficiently reduce ‘Nanog’ expression to 3% of control levels. By increasing the number of shRNA-mir hairpins against ‘Nanog’, efficiency of knockdown was further enhanced to nondetectable levels. Furthermore inducible knockdown of two other factors involved in ES cell pluripotency, was also achieved by using triple-hairpin shRNA-mir vectors, producing a phenotype consistent with previously reported shRNA experiments. Most attractive, the demonstrated multi-shRNA-mir strategy may be adapted to simultaneously reduce the expression of multiple, distinct target genes not only in ES cells, but also in vivo upon blastocyst injection of the shRNA-mir ES cells.

20.03.2008

The genetic design of signalling cascades to record receptor activation

Barnea, G. et al. 2008 PNAS 105: 64-69

Comment:

In a recent publication in PNAS, researchers from Sentigen Biosciences in collaboration with R. Axel’s laboratory at Columbia University, established a novel system to monitor protein interactions which was applied to GPCRs, receptor tyrosine kinases and steroid hormone receptors.
A fusion protein consisting of a membrane receptor and a Tet-regulated activator tethers Tet-Off to the cell membrane. Sequences of both proteins are separated by a linker containing a protease cleavage site. A second fusion protein links a viral protease to a cellular protein that interacts only with the activated receptor. Upon ligand binding to the receptor the protease is recruited to its cleavage site and will release the Tet-off transcription factor which subsequently will activate reporter gene expression. This so-called ‘Tango- assay’ converts the transient response of ligand binding to a receptor into a stable and easily quantifiable reporter gene signal thus allowing to record receptor activation without interference from endogenous signaling pathways.
The assay was developed for the three different types of receptors mentioned above and further on was shown to allow identification of endogenous ligands for orphan receptors as exemplified for GPR1.

29.03.2007

Organ size is limited by the number of embryonic progenitor cells in the pancreas but not the liver.

Stanger BZ et al. 2007 Nature 445:886-891

Comment:

Understanding of the mechanisms and key determinants that control vertebrate organ size is rather limited. In a recent publication in Nature Stanger et al. elegantly demonstrate that in the pancreas the number of progenitor cells determines final organ size rather than endocrine factors or extrinsic signals. Using transgenic mice expressing diphtheria toxin (DTA) under Tet-control they specifically destroyed pancreatic progenitor cells at distinct time points during embryogenesis. Newborn mice with progenitor cell ablation during the critical embryonic period showed nearly complete absence of the pancreas. Furthermore when the number of progenitor cells was reduced due to DTA expression during a brief period early in development prenatal embryos reproducibly had significantly smaller pancreata, indicating that the remaining progenitor cells were unable to compensate for the reduced cell number. Performing similar experiments in mice containing Tet regulated DTA expression directed specifically to hepatic progenitor cells demonstrated that in the liver growth compensation occurs rapidly even after sever reduction of hepatic progenitor cell number.
Thus while the number of progenitor cells is a critical determinant of pancreas size, liver size determination relies on extrinsic factors independent from progenitor cell pool size.

07.09.2006

Dissecting self-renewal in stem cells with RNA interference.

Ivanova N. et al; Nature. 2006 Jun 11

Comment:

In one of the most recent applications of the Tet-System, scientists at Princeton University identified key regulators of stem cell renewal and differentiation in mouse ES cells. Starting from a large number of down regulated genes detected in a microarray screening approach on mouse ES cells undergoing retinoic acid induced differentiation they focus on candidates suspected to control stem cell fate. Transduction of murine ES cells with lentiviral vectors encoding constitutively expressed shRNAs directed against each of the suspected regulators was then performed to measure ES cell differentiation in a fluorescence-based competition assay. Down regulation of several target messenger RNAs was associated with morphological changes and loss of alkaline phosphatase activity, indicative of a differentiated phenotype. In order to provide functional evidence for involvement of the suspected regulators in self-renewal of ES cells a genetic complementation strategy (rescue) was devised. For this purpose, the original shRNA lentiviral vectors were modified to allow Tet-induced expression of the shRNA-targeted candidates. Transduction of Tet-On-expressing ES cells with suspected regulators under Tet control revealed that self-renewal became doxycycline dependent suggesting specific biological roles as potent regulators of stem cell renewal for seven of the identified genes.