Summary Molecular mechanisms of development and disease can be studied in transgenic animals. Controlling the spatial and temporal expression patterns of transgenes, however, is a prerequisite for the elucidation of gene function in the whole organism. Mice carrying a tetR/VP16 hybrid gene (tTA) under the control of the human cytomegalovirus immediate early 1 (HCMV-IE1) gene promoter can be used to temporally activate the expression of transgenes under the control of a tetop promoter. In addition, the MMTV-LTR can be used to target expression of tTA to the epithelial cells of secretory organs and skin in transgenic mice. Notably, nearly uniform expression of a tetop-lacZ transgene was found in seminal vesicle, salivary gland and Leydig cells of mice carrying also the MMTV-tTA gene. More heterogeneous patterns of gene expression were observed in mammary epithelial cells and basal cells of the epidermis. Different MMTV-tTA lines had comparable tissue expression patterns. Transcriptional activation mediated by tTA was up to a/several thousand fold, and it was abrogated after the administration of tetracycline. The MMTV-tTA mice established were also used to examine the roles of biological factors at defined developmental stages in the epithelial cells of salivary gland, seminal vesicle, mammary gland and skin, and the Leydig cells of testes. In addition, in combination with the CRE/lox recombination system, these mice will be useful to achieve gene deletions at defined timepoints in these organs.
Background Dominant gain of function experiments in transgenic animals have been useful to elucidate the role of mammalian gene products in development and oncogenesis. Although it is possible to direct the expression of transgenes to defined tissues by employing specific genetic regulatory elements, conditional control of gene expression has been a challenge. For example, the promoter of the whey acidic protein (WAP) gene can target gene expression to mammary alveolar cells, but its temporal activation during puberty and pregnancy cannot be controlled by experimental conditions. The inability to directly control the temporal expression of transgenes has important consequences for transgenic studies designed to elucidate the role of proteins involved in development. The phenotype observed frequently will reflect the stage of development at which the transgene encoding the regulatory protein was first activated. For example, if expression of the transgene encoded protein causes lethality at a defined stage of development, it will not be possible to study its effect on later developmental stages. This is particularly important in cases in which a protein can play different roles which depend on the stage of development and the cell type. For example, depending on the time of expression during pregnancy, TGF-b can interfere with either ductal or alveolar development.
The development of regulatory circuits based on the tetracycline resistance operon tet from E.coli transposon Tn10 opened a new approach to controlling gene expression in eukaryotic cells. In this system, a transactivator protein (tTA) composed of the tet repressor and the activating domain of viral protein VP16 of herpes simplex virus activates transcription from a minimal promoter fused to seven tet operator sequences from Tn10 (tetop ). When an HCMVIE1 enhancer/promoter is used to direct tTA expression, tetop controlled target gene expression is activated in several different cell types although the strongest activation is observed in muscle cells (our paper). Transcriptional activation can be repressed by administration of tetracycline to the animal.
Numerous studies of the transforming potential of oncogenes and growth factors in transgenic mice have been focused on secretory tissues, such as the mammary gland, salivary gland and male reproductive organs. Conditional control over the expression of oncogenes and/or growth factors in these tissues would provide a new tool to study oncogenesis in these organs. For instance, oncogene and growth factor expression can be targeted to cells at specific stages of differentiation, the duration of oncoprotein or growth factor can be directly controlled, and expression of oncogenes or growth factors during embryogenesis can be avoided if desired. To establish a conditional gene expression system in the epithelial cells of different secretory tissues, we generated a hybrid gene composed of the MMTV-LTR and the coding region of the tTA transactivator protein (MMTV-tTA). The function of the MMTV-tTA hybrid gene was evaluated in both tissue culture cells and several different tissues of transgenic mice. Mice carrying this MMTV-tTA transgene were bred with mice transgenic for a tetop-b-galactosidase gene. The expression pattern in double transgenic MMTV-tTA/tetbn mice was examined to determine the cell type and number of expressing cells in the absence and presence of tetracycline.
Citations Hennighausen, L., Wall, R.J., Tillamnn, U., Li, M. and Furth, P.A. (1995). Conditional gene expression in secretory tissues and skin of transgenic mice using the MMTV-LTR and the tetracycline responsive system. J. Cell. Biochem. 59:463-472.
Gene expression
The transgenes were introduced into a Bl6xSJL background.
Beta-galactosidase activity was analyzed in tissues from mice that were transgenic for MMTV-LTR-tTA and tetop-nlacZ. Highly heterogenous expression was observed in mammary tissue.
Expression of the tetop-lacZ transgene was more homogenous in the salivary gland and in seminal vesicle.
Females were able to nurse and raise their litters. Whole mount and histology did not indicate any aberrant mammary development.
tet system, MMTV-LTR, lacZ, mammary gland, inducible gene expression
Lothar Hennighausen
National Institutes of Health
Bethesda, MD 20892
tel. 301-435-8907
(e-mail: mammary@nih.gov)
last update: June 1998 |
