Fkbp8: novel isoforms, genomic organization, and characterization of a forebrain promoter in transgenic mice

The immunophilin homolog FKBP8 has been implicated in the regulation of apoptosis. Here we show that the 38-kDa form of FKBP8 (FKBP38) derives from a truncated ORF. The extended FKBP8 ORFs are 46 and 44 kDa in mouse and 45 kDa in human. Although the genomic organization of mouse and human FKBP8 is evolutionarily conserved, additional first exons are encoded by the murine locus. A 4.4-kb murine Fkbp8 gene fragment, containing a GC-rich potential promoter, directed expression of a LacZ reporter gene to forebrain neurons in transgenic mice. Expression of the transgene was observed in CA1 pyramidal neurons of the hippocampus in transgenic mice from three lines. One transgenic founder mouse exhibited widespread forebrain expression of the LacZ transgene that resembles the pattern for the endogenous Fkbp8 gene. Thus promoter/enhancer elements for forebrain expression are located around the first exons of the mouse Fkbp8 gene.     

The specificity of the myelin basic protein gene promoter studied in transgenic mice.

The myelin basic proteins (MBPs) are a family of polypeptides that are predominantly expressed in the nervous system where they play a major role in myelination. We have generated four lines of transgenic mice carrying a transgene in which 1.34 kb of the 5'-flanking sequence of the mouse MBP gene was fused upstream of the coding region of the Escherichia coli lac Z gene in order to investigate developmental and tissue-specific expression of the MBP gene. Expression of both the lacZ transgene and the endogenous MBP gene followed a common developmental pattern in mouse brain. Transgene expression was detected in primary oligodendrocytes, but not in type 2 astrocytes. In addition, the lacZ gene product was expressed in epithelial cells of certain nonneural tissues, namely kidney, epididymis, ureter, and seminal vesicles. The ectopic expression of the transgene was associated with the development of DNase I hypersensitive sites at the site of insertion which was found to be within the intron 1 region of the endogenous MBP gene. The results reported here strongly suggest that the 1.34-kb 5'-flanking region of the MBP gene contains cis-regulatory elements that confer developmental regulation of the MBP gene, although this region appears to lack elements that restrict its expression to the nervous system.     

Enteroendocrine cell expression of a cholecystokinin gene construct in transgenic mice and cultured cells

CCK is predominantly expressed in subsets of endocrine cells in the intestine and neurons in the brain. We evaluated the expression of a CCK gene construct in transgenic mice and cultured cells to identify a genomic region that directs correct tissue- and cell-specific expression in enteroendocrine cells. The CCKL1 transgene contained 6.4 kb of mouse Cck fused to lacZ. Expression was evaluated in three transgenic lines (J11, J12, J14) by measurement of beta-galactosidase in tissue homogenates and frozen sections. Correct tissue-specific expression was observed, with beta-galactosidase activity detected in intestine and brain. However, there were differences seen in cell-specific expression in the intestine. Line J14 exhibited expression in CCK-endocrine cells, with expressing cells arising at the normal time during fetal development. However, transgene expression in line J12 intestine was limited to neurons of the enteric nervous system, which reflect an early fetal expression pattern for CCK. Analysis of an additional 15 transgenic founder mice demonstrated intestinal expression in 40% of transgenics, with expressing mice following either an endocrine cell pattern or a neuronal pattern in approximately equal numbers. CCKL1 transfection analysis in cultured cells also demonstrated enteroendocrine cell expression, with 100-fold enhanced activity in the enteroendocrine cell line STC-1 compared with nonendocrine cell lines. The results suggest that the minimal cis-regulatory DNA elements necessary for appropriate CCK expression in enteroendocrine cells reside within the 6.4-kb mouse genomic fragment.     

In vivo somatic delivery of plasmid DNA and retrograde transport to obtain cell-specific gene expression in the central nervous system

We utilised the retrograde transport machinery of neurones to deliver naked plasmid DNA into the central nervous system. A 5.4-kb fragment of the glycine receptor (GlyR) alpha1 subunit gene was cloned and used to drive the expression of a construct encoding for the enhanced green fluorescent protein (EGFP). Injections of the plasmid DNA in the tongue of mice resulted in the expression of the marker protein in hypoglossal motor neurones, showing that the GlyRalpha1 promoter sequence is sufficient to drive expression of the transgene. In order to determine the specificity of expression of the 5.4-kb fragment of the GlyR alpha1 subunit gene promoter, we subsequently injected the plasmid DNA into the mouse central nucleus of the amygdala. This nucleus receives projections from the parabrachial nucleus, a brainstem area that has a high density of GlyRs, and from the insular cortex, a forebrain structure devoid of GlyRs. We observed EGFP-labelled neurones in the parabrachial nucleus, but not in the insular cortex, indicating that the 5.4-kb GlyR alpha1 subunit gene promoter confers specificity of expression. This approach provides a simple and rapid way to identify, in vivo, promoter elements that mediate neurone-specific gene expression.     

Elucidation of the minimal sequence required to imprint H19 transgenes

The imprinted mouse H19 gene exhibits maternal allele-specific expression and paternal allele-specific hypermethylation. We previously demonstrated that a 14-kb H19 minitransgene possessing 5' differentially methylated sequence recapitulates the endogenous H19 imprinting pattern when present as high-copy arrays. To investigate the minimal sequences that are sufficient for H19 transgene imprinting, we have tested new transgenes in mice. While transgenes harboring limited or no 3' H19 sequence indicate that multiple elements within the 8-kb 3' fragment are required for appropriate imprinting, transgenes incorporating 1.7 kb of additional 5' sequence mimic the endogenous H19 pattern, including proper imprinting of low-copy arrays. One of these imprinted lines had a single 15.7-kb transgene integrant. This is the smallest H19 transgene identified thus far to display imprinting properties characteristic of the endogenous gene, suggesting that all cis-acting elements required for H19 imprinting in endodermal tissues reside within the 15.7-kb transgenic sequence.     

Separate cis-acting DNA elements of the mouse pro-alpha 1(I) collagen promoter direct expression of reporter genes to different type I collagen-producing cells in transgenic mice

The genes coding for the two type I collagen chains, which are active selectively in osteoblasts, odontoblasts, fibroblasts, and some mesenchymal cells, constitute good models for studying the mechanisms responsible for the cell-specific activity of genes which are expressed in a small number of discrete cell types. To test whether separate genetic elements could direct the activity of the mouse pro-alpha 1(I) collagen gene to different cell types in which it is expressed, transgenic mice were generated harboring various fragments of the proximal promoter of this gene cloned upstream of the Escherichia coli beta-galactosidase gene. During embryonic development, X-gal staining allows for the precise identification of the different cell types in which the beta-galactosidase gene is active. Transgenic mice harboring 900 bp of the pro-alpha 1(I) proximal promoter expressed the transgene at relatively low levels almost exclusively in skin. In mice containing 2.3 kb of this proximal promoter, the transgene was also expressed at high levels in osteoblasts and odontoblasts, but not in other type I collagen-producing cells. Transgenic mice harboring 3.2 kb of the proximal promoter showed an additional high level expression of the transgene in tendon and fascia fibroblasts. The pattern of expression of the lacZ transgene directed by the 0.9- and 2.3-kb pro-alpha 1(I) proximal promoters was confirmed by using the firefly luciferase gene as a reporter gene. The pattern of expression of this transgene, which can be detected even when it is active at very low levels, paralleled that of the beta-galactosidase gene. These data strongly suggest a modular arrangement of separate cell-specific cis-acting elements that can activate the mouse pro-alpha(I) collagen gene in different type I collagen-producing cells. At least three different types of cell- specific elements would be located in the first 3.2 kb of the promoter: (a) an element that confers low level expression in dermal fibroblasts; (b) a second that mediates high level expression in osteoblasts and odontoblasts; and (c) one responsible for high level expression in tendon and fascia fibroblasts. Our data also imply that other cis- acting cell-specific elements which direct activity of the gene to still other type I collagen-producing cells remain to be identified.     

A 5' differentially methylated sequence and the 3'-flanking region are necessary for H19 transgene imprinting.

The mouse H19 gene is expressed exclusively from the maternal allele. The imprinted expression of the endogenous gene can be recapitulated in mice by using a 14-kb transgene encompassing 4 kb of 5'-flanking sequence, 8 kb of 3'-flanking sequence, which includes the two endoderm-specific enhancers, and an internally deleted structural gene. We have generated multiple transgenic lines with this 14-kb transgene and found that high-copy-number transgenes most closely follow the imprinted expression of the endogenous gene. To determine which sequences are important for imprinted expression, deletions were introduced into the transgene. Deletion of the 5' region, where a differentially methylated sequence proposed to be important in determining parental-specific expression is located, resulted in transgenes that were expressed and hypomethylated, regardless of parental origin. A 6-kb transgene, which contains most of the differentially methylated sequence but lacks the 8-kb 3' region, was not expressed and also not methylated. These results indicate that expression of either the H19 transgene or a 3' DNA sequence is key to establishing the differential methylation pattern observed at the endogenous locus. Finally, methylation analysis of transgenic sperm DNA from the lines that are not imprinted reveals that the transgenes are not capable of establishing and maintaining the paternal methylation pattern observed for imprinted transgenes and the endogenous paternal allele. Thus, the imprinting of the H19 gene requires a complex set of elements including the region of differential methylation and the 3'-flanking sequence.     

A 5-kilobase pair promoter fragment of the murine epididymal retinoic acid-binding protein gene drives the tissue-specific, cell-specific, and androgen-regulated expression of a foreign gene in the epididymis of transgenic mice

The murine epididymis synthesizes and secretes a retinoic acid-binding protein (mE-RABP) that belongs to the lipocalin superfamily. The gene encoding mE-RABP is specifically expressed in the mouse mid/distal caput epididymidis under androgen control. In transgenic mice, a 5-kilobase pair (kb) promoter fragment, but not a 0.6-kb fragment, of the mE-RABP gene driving the chloramphenicol acetyltransferase (CAT) reporter gene restricted high level of transgene expression to the caput epididymidis. No transgene expression was detected in any other male or female tissues. Immunolocalization of the CAT protein and in situ hybridization of the corresponding CAT mRNA indicated that transgene expression occurred in the principal cells of the mid/distal caput epididymidis, thereby mimicking the spatial endogenous mE-RABP gene expression. Transgene and mE-RABP gene expression was detected from 30 days and progressively increased until 60 days of age. Castration, efferent duct ligation, and hormone replacement studies demonstrated that transgene expression was specifically regulated by androgen but not by any other testicular factors. Altogether, our results demonstrate that the 5-kb promoter fragment of the mE-RABP gene contains all of the information required for the hormonal regulation and the spatial and temporal expression of the mE-RABP gene in the epididymis.     

Tissue-specific and hormonally regulated expression of the puromycin N-acetyltransferase-encoding gene under control of the rabbit uteroglobin promoter in transgenic mice.

Transgenic mice bearing two fragments of the rabbit uteroglobin 5'-flanking region fused to the new reporter gene (pac) encoding puromycin N-acetyltransferase (PAC) showed a different pattern of expression. Transgenic lines (C0.4) harboring a 404-bp fragment (-396/+8) had a uterus-specific expression slightly inducible by estrogen, lacking detectable expression in other tissues where the uteroglobin-encoding gene is naturally expressed in rabbit. Transgenic lines (C3.2) bearing a longer fragment of 3.2-kb (-3254/+8) showed hormonally regulated expression in the uterus and the male genital tract, and detectable expression in the lung. In addition, the nonstimulated uterine expression of the transgene was higher in C0.4 lines than in C3.2 lines. It could be concluded that all sequences required for uterus-specific expression should be present within the 404-bp fragment, and that other upstream (-396) sequences are responsible for expression in the lung and male genital tract, as well as for a possible down modulation of expression in the uterus.     

Organ-specific expression of the lacZ gene controlled by the opsin promoter after intravenous gene administration in adult mice

BACKGROUND: The tissue-specific expression of an exogenous gene, under the influence of a tissue-specific promoter, has been examined in the past with pro-nuclear injections of the transgene and the development of transgenic mouse models. 'Adult transgenics' is possible with the acute expression of an exogenous gene that is administered to adult animals, providing the transgene can be effectively delivered to distant sites following an intravenous administration. METHODS: The organ specificity of exogenous gene expression in adult mice was examined with a bacterial beta-galactosidase (LacZ) expression plasmid under the influence of the bovine rhodopsin gene promoter. The 8-kb plasmid DNA was delivered to organs following an intravenous administration with the pegylated immunoliposome (PIL) non-viral gene transfer technology. The PIL carrying the gene was targeted to organs with the rat 8D3 monoclonal antibody (MAb) to the mouse transferrin receptor (TfR). RESULTS: The rhodopsin/beta-galactosidase gene was expressed widely in both the eye and the brain of adult mice, but was not expressed in peripheral tissues, including liver, spleen, lung, or heart. Ocular expression included the retinal-pigmented epithelium, the iris, and ciliary body, and brain expression was observed in neuronal structures throughout the cerebrum and cerebellum. CONCLUSIONS: The expression of trans-genes in adult animals is possible with the PIL non-viral gene transfer method. The opsin promoter enables tissue-specific gene expression in the eye, as well as the brain of adult mice, whereas gene expression in peripheral tissues, such as liver or spleen, is not observed.     

Expression of human IFN-gamma genomic DNA in transgenic mice

We have introduced an 8.6-kb fragment of human genomic DNA containing the full length IFN-gamma gene into the mouse germline. The transgenic animals had no biologic or developmental defects as human IFN-gamma does not bind to the mouse IFN R. Regulation of the transgene paralleled that of the endogenous murine IFN-gamma gene in that: 1) it is not expressed constitutively in any tissue examined thus far, 2) it can be induced in thymus and spleen cells by T cell mitogens, 3) it is not expressed in B cells stimulated by LPS, and 4) it produces normal mRNA and biologically active IFN protein. Whereas expression of the transgene is likely restricted to T cells, we had observed that both fibroblasts and B cell lines could express the same DNA when transfected in vitro; this indicates that in vivo, developmental factors restrict expression of the IFN-gamma gene to T cells. These findings also indicate that the 8.6-kb fragment contains the regulatory elements necessary for normal tissue specific expression in vivo. Moreover, they indicate that the regulatory elements for this gene are completely preserved over the phylogenetic distance separating mouse and man, even though substantial drift has occurred in the structural gene, and probably in the IFN-gamma R as well.