MISSION LentiPlex pooled shRNA library screening in mammalian cells.

RNA interference (RNAi) is an intrinsic cellular mechanism for the regulation of gene expression. Harnessing the innate power of this system enables us to knockdown gene expression levels in loss of gene function studies. There are two main methods for performing RNAi.

RNAi screen for telomerase reverse transcriptase transcriptional regulators identifies HIF1alpha as critical for telomerase function in murine embryonic stem cells.

In various types of stem cells, including embryonic stem (ES) cells and hematopoietic stem cells, telomerase functions to ensure long-term self-renewal capacity via maintenance of telomere reserve. Expression of the catalytic component of telomerase, telomerase reverse transcriptase (Tert), which is essential for telomerase activity, is limiting in many types of cells and therefore plays an important role in establishing telomerase activity levels. However, the mechanisms regulating expression of Tert in cells, including stem cells, are presently poorly understood.

Midline signaling regulates kidney positioning but not nephrogenesis through Shh.

The role of axial structures, especially the notochord, in metanephric kidney development has not been directly examined. Here, we showed that disruption of the notochord and floor plate by diphtheria toxin (DTA)-mediated cell ablation did not disrupt nephrogenesis, but resulted in kidney fusions, resembling horseshoe kidneys in humans. Axial disruptions led to more medially positioned metanephric mesenchyme (MM) in midgestation.

Cre/lox recombination in the lower urinary tract.

Tbx18 is a T-Box transcription factor that has specific expression and indispensible function in the lower urinary tract. Here, we report the generation and characterization of a bacterial artificial chromosome (BAC) transgene expressing Cre under the control of Tbx18 regulatory elements. When crossed to the ROSA26R-lacZ reporter mice, the Tbx18-Cre transgene mediates loxP recombination in the mesenchymal derivatives in the lower urinary tract, especially in the smooth muscle cells (SMCs) and the stromal cells.

Sirt1 deficiency attenuates spermatogenesis and germ cell function.

In mammals, Sirt1, a member of the sirtuin family of proteins, functions as a nicotinamide adenine dinucleotide-dependent protein deactylase, and has important physiological roles, including the regulation of glucose metabolism, cell survival, and mitochondrial respiration. The initial investigations of Sirt1 deficient mice have revealed a phenotype that includes a reduced lifespan, small size, and an increased frequency of abnormal sperm. We have now performed a detailed analysis of the molecular and functional effects of Sirt1 deficiency in the germ line of Sirt1 knock-out (-/-) mice.

SIRT1 acts as a nutrient-sensitive growth suppressor and its loss is associated with increased AMPK and telomerase activity.

SIRT1, the mammalian homolog of SIR2 in Saccharomyces cerevisiae, is an NAD-dependent deacetylase implicated in regulation of lifespan. By designing effective short hairpin RNAs and a silent shRNA-resistant mutant SIRT1 in a genetically defined system, we show that efficient inhibition of SIRT1 in telomerase-immortalized human cells enhanced cell growth under normal and nutrient limiting conditions. Hematopoietic stem cells obtained from SIRT1-deficient mice also showed increased growth capacity and decreased dependency on growth factors.

Regulation and effects of modulation of telomerase reverse transcriptase expression in primordial germ cells during development.

Telomere length maintenance in the germ line from generation to generation is essential for the perpetuation of eukaryotic organisms. This task is performed by a specialized reverse transcriptase called telomerase. While this critical function of telomerase has been well established, the mechanisms that regulate telomerase in the germ line are still poorly understood.

Structure of the bovine natural resistance associated macrophage protein (NRAMP 1) gene and identification of a novel polymorphism.

The NRAMP 1 gene is a major candidate gene influencing the outcome of infections with intracellular pathogens in numerous species. NRAMP 1 is highly conserved in many mammalian species and the NRAMP 1 gene shows considerable conservation in structure between mice and humans. The association of NRAMP 1 gene polymorphisms with disease in cattle has been limited to a single microsatellite located within the 3'-non coding region of the bovine NRAMP 1 gene.

Development and testing of a high-density cDNA microarray resource for cattle.

A cDNA microarray resource has been developed with the goal of providing integrated functional genomics resources for cattle. The National Bovine Functional Genomics Consortium's (NBFGC) expressed sequence tag (EST) collection was established in 2001 to develop resources for functional genomics research. The NBFGC EST collection and microarray contains 18,263 unique transcripts, derived from many different tissue types and various physiologically important states within these tissues.