Development of a unique system for spatiotemporal and lineage-specific gene expression in mice

Abstract

We have developed an advanced method for conditional gene expression in mice that integrates the Cre-mediated and tetracycline-dependent expression systems. An rtTA gene, preceded by a loxP-flanked STOP sequence, was inserted into the ROSA26 locus to create a R26STOPrtTA mouse strain. When the STOP sequence is excised by Cre-mediated recombination, the rtTA is expressed in the Cre-expressing cells and all of their derivatives. Therefore, cell type-, tissue-, or lineage-specific expression of rtTA is achieved by the use of an appropriate Cre transgenic strain. In mice also carrying a target gene under the control of the tetracycline response element, inducible expression of the target gene is temporally regulated by administration of doxycycline. Our results demonstrate that this universal system is uniquely suited for spatiotemporal and lineage-specific gene expression in an inducible fashion. Gene expression can be manipulated in specific cell types and lineages with a flexibility that is difficult to achieve with conventional methods.conditonal gene expression ͉ Cre ͉ ROSA26 locus ͉ rtTA D evelopment of advanced transgenic systems for conditional gene expression is essential for studying development and modeling human diseases in mice. Two powerful systems that have been used extensively are the Cre-loxP system, which allows genes to be permanently activated or inactivated in specific lineages (1), and the tetracycline-dependent system, which permits temporal as well as cell type-or tissue-specific control of transgene expression (2). The basic strategy for the Cre-loxP system is to delete the DNA sequence flanked by two loxP sites, thereby achieving conditional gene activation and inactivation. Manipulating the expression of Cre in a specific place and at a specific time enables conditional regulation. In the tetracyclinedependent system, three essential elements are required. First is the transcription factor tTA or rtTA, which can be expressed in a spatial specific manner. Second, the tetracycline response element (TRE) is used to control the expression of target genes. The presence of the third element, doxycycline (Dox), which can be administrated in a temporal specific fashion, activates target gene expression. We have developed a mouse strain, R26STOPrtTA, which combines features of these two systems and permits greater flexibility of conditional gene expression in the mouse. An rtTA gene, preceded by a loxP-flanked STOP sequence, was inserted into the ubiquitously active ROSA26 locus. When R26STOPrtTA is crossed with Cre-expressing strains, the STOP sequence is excised, yielding the R26rtTA allele, which expresses rtTA in the Cre-expressing cells and all of their descendants. Thus, the cell type, tissue, or lineage specificity of rtTA expression is dictated by selection of an appropriate Cre transgenic strain. In mice also carrying a target gene under the control of TRE, expression of the target gene is temporally regulated by administration of Dox. Using this approach, we have successfully achieved tr...

Genetic inducible fate mapping in mouse: Establishing genetic lineages and defining genetic neuroanatomy in the nervous system

“…For these alleles, it will be necessary to express the blockers in an inducible manner from the population of cells marked with GIFM. One approach that has been developed is activation of tTA or rtTA from a loxP-STOP allele (Park et al, 2004;Belteki et al, 2005;Yu et al, 2005). The neural activity blocker would then be expressed from a tetO phenotyping responder allele.…”

Reciprocal regulation of Wnt and Gpr177/mouse Wntless is required for embryonic axis formation

“…For embryo genotyping, yolk sacs or embryonic materials recovered from paraffin sections were used in PCR analysis. Axin2 GFP strain permits inducible expression of GFP in the Axin2-expressing cells (39,40). Care and use of experimental animals described in this work comply with guidelines and policies of the University Committee on Animal Resources at the University of Rochester.…”

“…RNA probes (5,25) were generated to analyze the gene expression pattern by in situ hybridization (45). Histology, ␤-gal staining, immunostaining, immunoblot, protein precipitation, chromatin immunoprecipitation, and various DNA vectors used are described in the SI Methods (39,40,42,(45)(46)(47).…”

Sustained Neurog3 expression in hormone-expressing islet cells is required for endocrine maturation and function

“…S1 A and B). Production of tTA, as a surrogate of Neurog3 expression, was examined with the strictly tTA-dependent reporter line, TetO LacZ (15,16). At embryonic days (E) 12.5 and 16.5 and postnatal day 7, and in the absence of doxycycline (Dox; the presence of which inhibits tTA activity), Neurog3 tTA ;TetO LacZ pancreata expressed LacZ in a subset of pancreatic cells reminiscent of Neurog3-expressing cells or their descendents (Fig.…”