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A conditional null allele for VCAM-1 was generated in mice through a one step ES cell selection procedure by flanking the proximal promoter and exons 1 and 2 with loxP sites. The ES cells were used to create chimeric mice, which were then used to produce mice homozygous for the VCAM-1 conditional null, or floxed allele. Although the PGKneo cassette was retained in the promoter, the homozygous mice produced levels of VCAM-1 transcripts similar to that seen in wild-type mice. Homozygous VCAMflox/flox mice were mated to transgenic lines of mice expressing the cre gene under control of the murine platelet endothelial cell adhesion molecule-1 (PECAM-1) promoter. Surprisingly, the VCAMflox allele in all tissues examined from mice that inherited the cre-transgene had underwent complete excision of the floxed VCAM-1 sequences. The 'deleted' VCAM-1 allele (VCAMdel) was stably inherited, even in those mice that did not inherit the cre transgene, indicating the recombination occurs at an early stage of development prior to germ cell development. Thus the cre mice can be used for ubiquitous gene rearrangement in vivo. The data also suggest a novel simplified strategy for using the Cre/loxP system in vivo, in which a single ES cell and line of mice can be used to create mice carrying either a null or conditional null allele.
Transgenic mice containing one or more extra copies of the entire glucokinase (GK) gene locus were generated and characterized. The GK transgene, an 83-kilobase pair mouse genomic DNA fragment containing both promoter regions, was expressed and regulated in a cell-specific manner, and rescued GK null lethality when crossed into mice bearing a targeted mutation of the endogenous GK gene. Livers from the transgenic mice had elevated GK mRNA, protein, and activity levels, compared with controls, and the transgene was regulated in liver by dietary manipulations. The amount of GK immunoreactivity in hepatocyte nuclei, where GK binds to the GK regulatory protein, was also increased. Pancreatic islets displayed increased GK immunoreactivity and NAD(P)H responses to glucose, but only when isolated and cultured in 20 mM glucose, as a result of the hypoglycemic phenotype of these mice (Niswender, K. D., Shiota, M., Postic, C., Cherrington, A. D., and Magnuson, M. A. (1997) J. Biol. Chem. 272, 22604-22609). Together, these results indicate that the region of the gene from -55 to +28 kilobase pairs (relative to the liver GK transcription start site) contains all the regulatory sequences necessary for expression of both GK isoforms, thereby placing an upper limit on the size of the GK gene locus.
Many transgenes, particularly those comprising cDNA sequences fail to be expressed when they are introduced into transgenic mice. We have previously shown that this problem can be overcome in the mammary gland by co-integrating a poorly expressed cDNA transgene, comprising the sheep beta-lactoglobulin promoter, with the efficiently expressed, unmodified beta-lactoglobulin gene. In this report we demonstrate that the transcription of the beta-lactoglobulin gene is associated with this effect because co-integration with a non-transcribed beta-lactoglobulin gene fails to rescue expression. By contrast, co-integration with a translationally inactivated beta-lactoglobulin transgene does rescue the expression of the second gene, but without the co-production of beta-lactoglobulin protein.
The ovine beta-lactoglobulin gene is expressed efficiently and at high levels in the mammary gland of transgenic mice. In contrast, when this gene is linked to a second gene construct comprising a mammalian cDNA or a CAT reporter sequence it fails to be expressed in the majority of transgenic lines generated. We suggest that mammalian cDNAs and prokaryotic reporter sequences can serve as active foci for gene silencing in the mammalian genome.
The beta-type transforming growth factors (TGF beta) are potent inhibitors of cell proliferation. The mechanisms of TGF beta growth inhibition have been investigated. In skin keratinocytes, TGF beta 1 rapidly suppresses c-myc expression at the level of transcriptional initiation, and expression of c-myc was shown to be necessary for proliferation of these cells. Overexpression of c-myc, using an inducible construct, blocks growth inhibition by TGF beta 1. In 11.5 day p.c. lung bud organ cultures, TGF beta 1 inhibits tracheobronchial epithelial development, including branching morphogenesis. At this stage of development, the tracheobronchial epithelia express N-myc, but not c-myc, TGF beta 1 was shown to markedly inhibit N-myc expression in epithelia of the lung bud organ cultures. N-myc gene knockout experiments by others have shown that N-myc is required for branching morphogenesis of the tracheobronchial tree. The data indicate that suppression of expression of either N-myc or c-myc may play a role in TGF beta growth inhibition. To study the role of TGF beta 1 in normal mammary development and in mammary neoplasia, we have constructed three transgenic mouse lines that express a simian TGF beta 1S223/225 mutated to produce a constitutively active product under the control of the MMTV enhancer/promoter. Expression of the transgene was associated with marked suppression of the normal pattern of mammary ductal tree development in female transgenics from all three lines. However, during pregnancy, alveolar outgrowths developed from the hypoplastic ductal tree, and lactation occurred. Unlike many other transgenic mouse models in which expression of TGF alpha or oncogenes under control of the MMTV promoter leads to mammary epithelial hyperplasia and increased tumor formation, the MMTV-TGF beta 1 transgene causes conditional hypoplasia of the mammary ductal tree. No spontaneous tumors have been detected in the MMTV-TGF beta 1 transgenic animals, indicating that overexpression of TGF beta 1 in mammary epithelia does not enhance, and may actually suppress, early stages of carcinoma development. Other studies have shown that overexpression of TGF beta 1 in carcinoma cells enhances tumorigenicity and metastatic spread. We propose that TGF beta has a bifunctional role in carcinogenesis, retarding carcinoma development but enhancing progression once neoplastic transformation has occurred and the growth inhibitory response to TGF beta has been lost.