The transcription factor E2F-1 in SV40 T antigen-induced cerebellar Purkinje cell degeneration.

Transgenic targeting of SV40 large T antigen (Tag) expression to murine cerebellar Purkinje cells induces these normally postmitotic neurons to undergo DNA synthesis and apoptosis. It has been proposed that these effects of Tag are due to the binding of Tag to pRb, which leads to the release and activation of the transcription factor E2F. Here it is reported that E2F and CDC2, the protein product of a gene regulated by E2F, were detectable in the Purkinje cell nuclei of Tag expressing transgenic animals. To directly test whether E2F-1 is part of the mechanism of Tag-induced Purkinje cell degeneration, transgenic mice that overexpress E2F-1 specifically in cerebellar Purkinje cells were generated. Although E2F-1 itself did not affect Purkinje cells, it did accelerate Tag-induced ataxia and Purkinje cell loss, suggesting that E2F-1 can contribute to the mechanism of Tag-induced Purkinje cell degeneration.

Isolation, characterization and in vivo analysis of the murine calbindin-D28K upstream regulatory region.

The genomic locus containing the murine calbindin-D28K gene has been isolated and partially characterized. Genomic cloning revealed an exon/intron chromosomal structure very similar to the avian gene previously described. The ability of the calbindin-D28K upstream region to direct cell-specific expression was tested in vivo. Varying lengths of upstream sequence were used to drive expression of lacZ in transgenic mice. Characterization of 23 transgenic mouse lines revealed that even as much as 3.0 kb of upstream sequence was unable to direct expression independently of integration site effects, suggesting the absence of important elements. Despite the small number of expressing transgenic lines and the great variability, there was a tendency of cell specificity of transgene expression exhibited in distinct brain regions. In the cerebellum, Purkinje cell-specific expression was observed with the shortest (1.0 kb) upstream sequence tested. Specificity of transgene expression in Purkinje cells was abolished with longer portions of upstream sequence. The same observation was made for transgene expression in granule cells of the dentate gyrus, while the opposite effect was observed for expression in CA1 hippocampal cells. The absence of any transgenic lines exhibiting appropriate transgene expression in the kidney suggested that the VDREs described previously for the murine calbindin gene are not sufficient to direct kidney expression in vivo. It is concluded that 3.0 kb of calbindin upstream sequence includes the regulatory elements dictating a portion of cell-specificity in the CNS of transgenic mice, albeit lacking regions that allow expression independently of chromosomal effects.

Temporal and spatial expression of HLA-G messenger RNA in extraembryonic tissues of transgenic mice.

HLA-G, a nonclassical class I molecule, is expressed by trophoblasts, the only fetal cells in direct contact with maternal tissue. Results of previous experiments suggested that a 244-bp region located over 1 kb 5' from exon 1 is critical for extraembryonic expression of HLA-G in transgenic mice. We report here the production of HLA-G transgenic lines with a 6.0-kb HLA-G transgene that includes the 244-bp region. These lines exhibit copy number-dependent and developmentally appropriate transgene expression. One HLA-G transgenic line, G.3.2, exhibits extraembryonic HLA-G mRNA expression levels similar to those seen in human extraembryonic tissues. Studies of the cell-type-specific localization of HLA-G mRNA in placentas from the G.3.2 HLA-G transgenic mouse line show predominant localization of the HLA-G message in the spongiotrophoblast layer. This layer is in a similar anatomic location to the HLA-G-expressing human cytotrophoblast shell. The G.3.2 HLA-G transgenic mouse line should serve as an appropriate model for the study of HLA-G function at the maternal-fetal interface.

Cellular distribution of HLA-G mRNA in transgenic mouse placentas.

The human MHC class I gene, HLA-G, is unique among members of the class I gene family in that it is nonpolymorphic, and expression is primarily restricted to extraembryonic tissues. To examine regulatory elements that direct tissue- and cell lineage-specific expression of this gene, transgenic mice expressing HLA-G have been established. In this study, in situ hybridization was used to evaluate the cellular distribution of HLA-G mRNA in transgenic placentas. Extraembryonic tissues were obtained at gestation day 12.5 from embryos that had been microinjected with either 6.0 or 5.7 kb of HLA-G genomic DNA and had been transferred into pseudopregnant HLA-G transgenic mice or Swiss mice. The 6.0 kb transgene contained an additional 250 bp at the extreme 5'-end of the upstream region. Genotype of the recipient had no discernable effect on the cellular distribution of HLA-G mRNA. HLA-G mRNA was present in both trophoblast and mesenchymal cells in transgenic placentas carrying 6.0 kb of genomic HLA-G, a pattern strikingly similar to that of HLA-G message distribution in early gestation human placentas. By contrast, in placentas from embryos carrying 5.7 kb HLA-G DNA, specific mRNA was found primarily in mesenchymal cells at the base of the placenta. Thus, the 6.0 genomic fragment contains elements capable of directing HLA-G expression in placentas, and is particularly influential in the trophoblastic cell lineage.

Extraembryonic expression of the human MHC class I gene HLA-G in transgenic mice. Evidence for a positive regulatory region located 1 kilobase 5' to the start site of transcription.

Trophoblast, the only fetal tissue in direct contact with maternal cells, fails to express the polymorphic HLA class I molecules HLA-A and -B, but does express the nonpolymorphic class I molecule HLA-G. It is thought that HLA-G may provide some of the functions of a class I molecule without stimulating maternal immune rejection of the fetal semiallograft. As a first step in identifying the cis-acting DNA regulatory elements involved in the control of class I expression by extraembryonic tissue, several types of transgenic mice were produced. Two HLA-G genomic fragments were used, 5.7 and 6.0 kb in length. These included the entire HLA-G coding region, 1 kb of 3' flanking sequence, and 1.2 or 1.4 kb of 5' flanking sequence, respectively. A hybrid transgene, HLA-A2/G, was produced by replacing the 5' flanking sequence, first exon, and early first intron of HLA-G with the corresponding elements of HLA-A. Comparison of transgene mRNA expression patterns seen in HLA-A2/G and HLA-G transgenic mice suggests that 5' flanking sequences are largely responsible for the differing patterns of expression typical of the classical class I and HLA-G genes. Studies comparing the extraembryonic HLA-G expression levels of founder embryos transgenic for either the 5.7- or 6.0-kb HLA-G transgene showed that the 6.0-kb transgene directed HLA-G expression far more efficiently than did the 5.7-kb HLA-G transgene, producing extraembryonic HLA-G mRNA levels similar to those seen in human extraembryonic tissues. The results of these studies suggest that the 250-bp fragment present at the extreme 5' end of the 6.0-kb HLA-G transgene and absent from the 5.7-kb HLA-G transgene contains an important positive regulatory element. This 250-bp fragment lies further upstream than any of the previously documented class I regulatory regions and may function as a locus control region.

Isoproterenol/tannin-dependent R15 expression in transgenic mice is mediated by an upstream parotid control region.

Transgenic mice were used to locate the cis-acting DNA elements that are essential for tissue-specific and inducible expression of the rat proline-rich protein gene, R15. Chimeric genes with up to 10 kb of R15 5'-flanking region fused to chloramphenicol acetyltransferase (CAT) or polyomaviral large T-antigen (PyLT) reporter genes were tested. Our results demonstrate that (1) the isoproterenol/tannin-inducible, parotid-specific transgene expression requires an upstream cis-regulatory domain, namely the parotid control region, which extends from -6 to -1.7 kb of the R15 gene; (2) this parotid control region functions with a heterologous promoter and is indispensable for achieving a reproducible chromosomal position-independent transgene expression; (3) deletion of the R15 5'-flanking region up to -1.7 kb results in a pleiotropic effect on the transgene expression, which includes ectopic (nonsalivary) reporter expression and lack of inducibility by either the beta-agonist isoproterenol or dietary tannin stimulation; (4) when the -10 to -6 kb region from the R15 gene is deleted in the construct, the inducible expression in the parotid glands of the transgenic mice decreases by over 30-fold, but position-independent and tissue-specific transgene expression is retained. Moreover, the mechanism of induction by either catecholamine isoproterenol or dietary tannin appears to be through a beta 1-adrenergic receptor-mediated pathway for both normal (non-transgenic) and transgenic animals.

Disrupted cerebellar cortical development and progressive degeneration of Purkinje cells in SV40 T antigen transgenic mice.

SV40 T antigen (Tag) expression directed to cerebellar Purkinje cells resulted in the generation of three transgenic mouse lines that displayed ataxia, a neurological phenotype characteristic of cerebellar dysfunction. Onset of symptoms and cerebellar pathology, characterized by specific Purkinje cell degeneration, appeared to be directly dependent upon transgene copy number. The SV5 line (containing > 30 transgene copies), exhibited embryonic transgene expression that caused selective death of immature Purkinje cells and a subsequent block in cerebellar development and ataxia at 2 weeks. The developmental effect of the disruption of Purkinje cells in SV5 mice suggests that a normal complement of these cells is required for early development of the cerebellar cortex, especially granule cell proliferation and migration from external to internal layers. Transgene expression in a second line, SV4 (10 copies), was detectable during the second postnatal week. Death of mature Purkinje cells in the SV4 line resulted in onset of ataxia at 9 weeks. Ataxia in a third line, SV6 (2 copies), was detected after 15 weeks. The distinct cerebellar phenotypes of the SV4-6 lines correlate with specific Tag-induced Purkinje cell ablation as opposed to tumorigenesis.