SV40 T antigen directed by a powerful erythroid enhancer-promoter produced sarcomas and pancreatic tumors but not erythroid-specific tumors in transgenic mice

We have expressed the simian virus 40 (SV40) large T antigen oncogene in erythroid tissues of mice to test its ability to immortilize erythroid cells. A transgene construct was built in which the SV40 large T antigen structural gene was linked to erythroid-specific enhancer and promoter sequences. The enhancer employed was the human beta-globin family microlocus control region, and the promoter sequences were derived from the human beta-globin promoter. Transgenic mice were generated and they expressed T antigen in the bone marrow and spleen cells. Yet, no hematopoietic neoplasia arose in these mice. Instead, after a lag period of 2-6 months, the mice developed soft tissue sarcomas and pancreatic islet-cell tumors that expressed high levels of T antigen.     

Substitution of the human beta-spectrin promoter for the human agamma-globin promoter prevents silencing of a linked human beta-globin gene in transgenic mice

During development, changes occur in both the sites of erythropoiesis and the globin genes expressed at each developmental stage. Previous work has shown that high-level expression of human beta-like globin genes in transgenic mice requires the presence of the locus control region (LCR). Models of hemoglobin switching propose that the LCR and/or stage-specific elements interact with globin gene sequences to activate specific genes in erythroid cells. To test these models, we generated transgenic mice which contain the human Agamma-globin gene linked to a 576-bp fragment containing the human beta-spectrin promoter. In these mice, the beta-spectrin Agamma-globin (betasp/Agamma) transgene was expressed at high levels in erythroid cells throughout development. Transgenic mice containing a 40-kb cosmid construct with the micro-LCR, betasp/Agamma-, psibeta-, delta-, and beta-globin genes showed no developmental switching and expressed both human gamma- and beta-globin mRNAs in erythroid cells throughout development. Mice containing control cosmids with the Agamma-globin gene promoter showed developmental switching and expressed Agamma-globin mRNA in yolk sac and fetal liver erythroid cells and beta-globin mRNA in fetal liver and adult erythroid cells. Our results suggest that replacement of the gamma-globin promoter with the beta-spectrin promoter allows the expression of the beta-globin gene. We conclude that the gamma-globin promoter is necessary and sufficient to suppress the expression of the beta-globin gene in yolk sac erythroid cells.     

Differential regulation of coproporphyrinogen oxidase gene between erythroid and nonerythroid cells

Coproporphyrinogen oxidase (CPO) catalyzes the sixth step of the heme biosynthetic pathway. To assess the tissue-specific regulation of the CPO gene promoter, mouse genomic DNA clones for CPO were isolated. Structural analysis demonstrated that the mouse CPO gene spans approximately 11 kb and consists of seven exons, just like its human counterpart. Functional analysis of the promoter by transient transfection assays indicated that synergistic action between an SP-1-like element at -21/-12, a GATA site at -59/-54, and a novel regulatory element, CPRE (-GGACTACAG-) at -49/-41, is essential for the promoter activity in murine erythroleukemia (MEL) cells. In nonerythroid NIH3T3 cells, however, the GATA site is not required. Gel mobility shift assays demonstrated that specific DNA-protein complexes can be formed with each element, and that there are cell-specific differences in factors, which bind to the SP-1-like element between MEL and NIH3T3 cells. These results provide evidence for differential regulation of the promoter function of CPO gene between erythroid and nonerythroid cells.     

Position independent expression and developmental regulation is directed by the beta myosin heavy chain gene's 5' upstream region in transgenic mice

Transgenic mice generated with constructs containing 5.6 kb of the beta myosin heavy chain (MyHC) gene's 5' flanking region linked to the cat reporter gene express the transgene at high levels. In all 47 lines analyzed, tissue-specific accumulation of chloramphenicol acetyltransferase was found at levels proportional to the number of integrated transgene copies. Deletion constructs containing only 0.6 kb of 5' upstream region showed position effects in transgenic mice and did not demonstrate copy number dependence although transgene expression remained muscle-specific. The 5.6 kb 5' upstream region conferred appropriate developmental control of the transgene to the cardiac compartment and directs copy number dependent and position independent expression. Lines generated with a construct in which three proximal cis-acting elements were mutated showed reduced levels of transgene expression, but all maintained their position independence and copy number dependence, suggesting the presence of distinct regulatory mechanisms.     

Organization of the proximal promoter of the hatching-enzyme gene, the earliest zygotic gene expressed in the sea urchin embryo

The hatching enzyme (HE) gene is the earliest zygotic gene expressed in the sea urchin embryo. To investigate the regulation of the HE gene activity, 5' flanking DNA and the 5' untranslated leader were inserted upstream of reporter genes whose expression was monitored in vivo during development after transfer into eggs. By deletion analysis we showed that no more than 3 kb of flanking sequence are required for correct expression of transgenes. The proximal region of 0.5 kb does not precisely control spatial restriction but drives expression at a nearly maximal level. The proximal promoter was searched extensively for sites of protein-DNA interactions by DNAse protection and gel-shift methods. The 12 sites identified form 3 groups: core promoter; central region; and distal region. The central region bears three sites that contain a direct or inverted CCAAT box. Mutation and deletion analysis showed that, in addition to the core-promoter elements, the two most-distal CCAAT-containing sites are indispensable for promoter activity. These sites bind the same set of proteins, which are abundant in the nuclei of cleavage embryos.     

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.     

Purification and nucleic-acid-binding properties of a Saccharomyces cerevisiae protein involved in the control of ploidy

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 6.1 kb 5' upstream region of the mouse tryptophan hydroxylase gene directs expression of E. coli lacZ to major serotonergic brain regions and pineal gland in transgenic mice

Tryptophan hydroxylase (TPH) catalyzes the first step of serotonin biosynthesis in serotonergic neurons and neuroendocrine cells. Serotonin influences diverse vital physiological functions and is thought to play an important role in several human psychiatric disorders. To localize DNA element(s) important for serotonergic tissue-specific expression of TPH, 6.1 kb of the 5' flanking region of the mouse TPH gene was fused to the coding region of the E. coli lacZ gene, and expression of the resulting fusion gene was analyzed in transgenic mice. The 6.1 kb of 5' flanking sequence was able to direct the expression of a lacZ reporter gene to serotonergic tissues in six lines of transgenic mice. A high level of lacZ expression in transgenic mice carrying the fusion gene was detected in the pineal gland as well as a moderate level of lacZ expression in serotonergic brain regions such as the median and dorsal raphe nuclei, the nuclei raphe magnus and raphe pallidus. In contrast, a smaller 5' flanking sequence of 1.1 kb directed no detectable serotonergic tissue-specific lacZ expression in five lines of transgenic mice. These results presented in this paper suggest first that DNA elements critical to serotonergic tissue-specific expression reside between -6.1 kb and -1.1 kb of 5' flanking region of the mouse TPH gene, but second that this region confers a restricted tissue-specific expression.