Ablation of a specific cell population by the replacement of a uniquely expressed gene with a toxin gene

The transgenic expression of a toxin gene or a thymidine kinase gene under the control of cell type-specific promoter/enhancer has been shown to be useful for removing a specific cell population in mice. However, this approach requires extensive analysis of the control elements for gene expression in the preparation of the transgenic constructs, and furthermore, the toxin gene might be expressed ectopically because of random integration, resulting in aberrant depletion of unrelated cells. To avoid such difficulties with the transgenic approach, we established a method for the specific depletion of a cell population by replacing a uniquely expressed gene in the population with the diphtheria toxin gene by using homologous recombination. The NKR-P1 gene, a specific cell surface marker of natural killer (NK) cells, was selected as the target gene for depleting NK cells. In chimeric mice reconstituted with embryonic stem cells in which the NKR-P1 gene was replaced by the toxin gene, NKR-P1(+) cells were almost completely depleted, and NK cell function was abrogated in the embryonic stem cell-derived lymphoid cells. Other cell lineages developed normally. These results show that all NK cells express NKR-P1, that NKR-P1(+) cells do not influence the development of T and B cells, and further, that this technology of cell targeting is a fast and powerful method of generating mice lacking any chosen cell population.     

Transgenesis and gene targeting in the mouse; tools for studying genetic determinants of hypertension

As more effort is made to identify genes responsible for hypertension in human populations and genetically hypertensive animal models, the need for experimental systems in which the functional significance of genes, gene variants, and quantitative trait loci (QTL) can be determined is becoming increasingly important. Over the past five years, transgenic and gene-targeting technology has been utilized to study the cardiovascular effects of over-expression or ablation of genes which have been considered candidates in the genetic basis of hypertension. This review focuses on the most recent major advances in this area, and how this technology aids in our understanding of the molecular mechanisms by which newly discovered genes or gene variants affect blood pressure in the whole organism. We also discuss the potential use of transgenic models in refining the location of a QTL, and discuss some of the limitations and potential pitfalls in the application of these tools to the field of hypertension research. The coupling of genetic manipulations afforded by transgenesis and gene targeting, along with advances in our ability to assess the cardiovascular phenotype in the mouse, provides us with a powerful system for examining the genes responsible for causing essential hypertension.     

Inducible and reversible gene silencing by stable integration of an shRNA-encoding lentivirus in transgenic rats

Currently, tools to generate loss-of-function mutations in rats are limited. Therefore, we have developed a lentiviral single-vector system for the temporal control of ubiquitous shRNA expression. Here, we report transgenic rats carrying an insulin receptor-specific shRNA transcribed from a regulatable promoter and identified by concomitant EGFP expression. In the absence of the inducer doxycycline (Dox), we observed no siRNA expression. However, Dox treatment at very low concentrations led to a rapid induction of the siRNA and ablation of INSR protein expression. As anticipated, blood glucose levels increased, whereas insulin signaling and glucose regulation were impaired. Importantly, this phenotype was reversible (i.e., discontinuation of Dox treatment led to INSR re-expression and remission of diabetes symptoms). The lentiviral system offers a simple tool for reversible gene ablation in the rat and can be used for other species that cannot be manipulated by conventional recombination techniques.     

Multiplex gene regulation: a two-tiered approach to transgene regulation in transgenic mice.

Transgenic mice have been used to study gene function and regulation by introducing inducible or tissue-specific transgenes. This approach is generally limited to studying gene function in adult mice since ectopic expression of many interesting genes is disease causing or may be lethal to the developing embryo. To extend the utility of the transgenic mouse system to the early stages of embryogenesis, we have developed a two-tiered method of gene regulation to control transgene expression. Our multiplex gene regulatory system (MGR) allows the establishment of transgenic lines that harbor inducible potentially lethal transgenes. These inducible transgenes are activated only when mated to a second transgenic animal. Induction in the MGR system provides a high degree of temporal and spatial control over transgene expression and should be suitable for engineering "gain of function mutations" for many developmental genes.