Promoter sequences targeting tissue-specific gene expression of hypothalamic and ovarian gonadotropin-releasing hormone in vivo.

Molecular mechanisms directing tissue-specific expression of gonadotropin-releasing hormone (GnRH) are difficult to study due to the paucity and scattered distribution of GnRH neurons. To identify regions of the mouse GnRH (mGnRH) promoter that are critical for appropriate tissue-specific gene expression, we generated transgenic mice with fragments (-3446/+23 bp, -2078/+23 bp, and -1005/+28 bp) of mGnRH promoter fused to the luciferase reporter gene. The pattern of mGnRH promoter activity was assessed by measuring luciferase activity in tissue homogenates. All three 5'-fragments of mGnRH promoter targeted hypothalamic expression of the luciferase transgene, but with the exception of the ovary, luciferase expression was absent in non-neural tissues. High levels of ovarian luciferase activity were observed in mice generated with both -2078 and -1005 bp of promoter. Our study is the first to define a region of the GnRH gene promoter that directs expression to both neural and non-neural tissues in vivo. We demonstrate that DNA sequences contained within the proximal -1005 bp of the mGnRH promoter are sufficient to direct mGnRH gene expression to both the ovary and hypothalamus. Our results also suggest that DNA sequences distal to -2078 bp mediate the repression of ovarian GnRH.     

A modified RMCE-compatible Rosa26 locus for the expression of transgenes from exogenous promoters

Generation of gain-of-function transgenic mice by targeting the Rosa26 locus has been established as an alternative to classical transgenic mice produced by pronuclear microinjection. However, targeting transgenes to the endogenous Rosa26 promoter results in moderate ubiquitous expression and is not suitable for high expression levels. Therefore, we now generated a modified Rosa26 (modRosa26) locus that combines efficient targeted transgenesis using recombinase-mediated cassette exchange (RMCE) by Flipase (Flp-RMCE) or Cre recombinase (Cre-RMCE) with transgene expression from exogenous promoters. We silenced the endogenous Rosa26 promoter and characterized several ubiquitous (pCAG, EF1α and CMV) and tissue-specific (VeCad, αSMA) promoters in the modRosa26 locus in vivo. We demonstrate that the ubiquitous pCAG promoter in the modRosa26 locus now offers high transgene expression. While tissue-specific promoters were all active in their cognate tissues they additionally led to rare ectopic expression. To achieve high expression levels in a tissue-specific manner, we therefore combined Flp-RMCE for rapid ES cell targeting, the pCAG promoter for high transgene levels and Cre/LoxP conditional transgene activation using well-characterized Cre lines. Using this approach we generated a Cre/LoxP-inducible reporter mouse line with high EGFP expression levels that enables cell tracing in live cells. A second reporter line expressing luciferase permits efficient monitoring of Cre activity in live animals. Thus, targeting the modRosa26 locus by RMCE minimizes the effort required to target ES cells and generates a tool for the use exogenous promoters in combination with single-copy transgenes for predictable expression in mice.     

An upstream regulatory region mediates high-level, tissue-specific expression of the human alpha 1(I) collagen gene in transgenic mice.

Studies in vitro have not adequately resolved the role of intronic and upstream elements in regulating expression of the alpha 1(I) collagen gene. To address this issue, we generated 12 separate lines of transgenic mice with alpha 1(I) collagen-human growth hormone (hGH) constructs containing different amounts of 5'-flanking sequence, with or without most of the first intron. Transgenes driven by 2.3 kb of alpha 1(I) 5'-flanking sequence, whether or not they contained the first intron, were expressed at a high level and in a tissue-specific manner in seven out of seven independent lines of transgenic mice. In most tissues, the transgene was expressed at levels approaching that of the endogenous alpha 1(I) gene and was regulated identically with the endogenous gene as animals aged. However, in lung, expression of the transgene was anomalously high, and in muscle, expression was lower than that of the endogenous gene, suggesting that in these tissues other regions of the gene may participate in directing appropriate expression. Five lines of mice were generated containing transgenes driven by 0.44 kb of alpha 1(I) 5'-flanking sequence (with or without the first intron), and expression was detected in four out of five of these lines. The level of expression of the 0.44-kb constructs in the major collagen-producing tissues was 15- to 500-fold lower than that observed with the longer 2.3-kb promoter. While transgenes containing the 0.44-kb promoter and the first intron retained a modest degree of tissue-specific expression, those without the first intron lacked tissue specificity and were poorly expressed in all tissues except lung. These results contribute to our understanding of the role of the first intron in regulating alpha1(I) gene expression and identify a region, upstream of the basal alpha1(I) promotor, which is necessary for full tissue-specific, developmentally regulated expression of the alpha1(I) collagen gene.     

Metabolic effects of developmental, tissue-, and cell-specific expression of a chimeric phosphoenolpyruvate carboxykinase (GTP)/bovine growth hormone gene in transgenic mice.

Transgenic mice were used to investigate sequences within the promoter of the gene for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) from the rat (EC 4.1.1.32) (PEPCK) which are involved in tissue-specific and developmental regulation of gene expression. Segments of the PEPCK promoter between -2000 and -109 were linked to the structural gene for bovine growth hormone (bGH) and introduced into the germ line of mice by microinjection. Bovine growth hormone mRNA was found in tissues that express the endogenous PEPCK gene, mainly in the liver but to a lesser extent in the kidney, adipose tissue, small intestine, and mammary gland. In the liver the chimeric PEPCK/bGH(460) gene was expressed in periportal cells, which is consistent with the zonation of endogenous PEPCK. The PEPCK/bGH gene was not transcribed in the livers of fetal mice until immediately before birth; at birth the concentration of bGH mRNA increased 200-fold. Our results indicate that the region of the PEPCK promoter from -460 to +73 base pairs contains regulatory sequences required for tissue-specific and developmental regulation of PEPCK gene expression. Mice transgenic for PEPCK/bGH(460) were not hyperglycemic or hyperinsulinemic in response to elevated bGH, as were transgenic mice with the MT/bGH gene. The number of insulin receptors in skeletal muscle was no different in mice transgenic for MT/bGH when compared with mice transgenic for PEPCK/bGH(460) and control animals. However, mRNA abundance for the insulin-sensitive glucose transporter in skeletal muscle was decreased in mice transgenic for the MT/bGH gene. The differences in glucose homeostasis noted with the two types of transgenic mice may be the result of the relative site of expression, the different developmental pattern, or hormonal regulation of expression of the bGH gene.     

BLG-e1 - a novel regulatory element in the distal region of the beta-lactoglobulin gene promoter

beta-Lactoglobulin (BLG) is a major ruminant milk protein. A regulatory element, termed BLG-e1, was defined in the distal region of the ovine BLG gene promoter. This 299-bp element lacks the established cis-regulatory sequences that affect milk-protein gene expression. Nevertheless, it alters the binding of downstream BLG sequences to histone H4 and the sensitivity of the histone-DNA complexes to trichostatin A treatment. In mammary cells cultured under favorable lactogenic conditions, BLG-e1 acts as a potent, position-independent silencer of BLG/luciferase expression, and similarly affects the promoter activity of the mouse whey acidic protein gene. Intragenic sequences upstream of BLG exon 2 reverse the silencing effect of BLG-e1 in vitro and in transgenic mice.     

Analysis of tissue-specific expression of human type II collagen cDNA driven by different sizes of the upstream region of the beta-casein promoter

To investigate the ability of 1.8 kb or 3.1 kb bovine beta-casein promoter sequences for the expression regulation of transgene in vivo, transgenic mice were produced with human type II collagen gene fused to 1.8 kb and 3.1 kb of bovine beta-casein promoter by DNA microinjection. Five and three transgenic founder mice were produced using transgene constructs with 1.8 kb and 3.1 kb of bovine beta-casein promoters respectively. Founder mice were outbred with the wild type to produce F1 and F2 progenies. Total RNAs were extracted from four tissues (mammary gland, liver, kidney, and muscle) of female F1 transgenic mice of each transgenic line following parturition. RT-PCR and Northern blot analysis revealed that the expression level of transgene was variable among the transgenic lines, but transgenic mice containing 1.8 kb of promoter sequences exhibited more leaky expression of transgene in other tissues compared to those with 3.1 kb promoter. Moreover, Western blot analysis of transgenic mouse milk showed that human type II collagen proteins secreted into the milk of lactating transgenic mice contained 1.8 kb and 3.1 kb of bovine beta-casein promoter. These results suggest that promoter sequences of 3.1 kb bovine beta-casein gene can be used for induction of mammary gland-specific expression of transgenes in transgenic animals.     

Characterization of recombinant human factor IX expressed in transgenic mice and in derived trans-immortalized hepatic cell lines.

Transgenic mice were generated in which 5 kb of the 5' flanking promoter region of the human Factor IX (FIX) gene fused to various FIX constructs (gene, minigene and cDNA) were stably integrated in the germ line. Several transgenic mouse lines expressed high circulating levels of active and correctly processed recombinant human FIX. The presence of at least one FIX intron had a positive effect on the expression. The FIX transgenes were expressed in a tissue-specific manner in the liver of transgenic mice. By crossing transgenic mice synthesizing FIX with others prone to develop hepatoma, progeny which co-express the transgenes in hepatocytes were obtained. Hepatoma-derived cell lines were shown to have a differentiated phenotype and secrete active human FIX for many generations.     

Retina-specific expression from the IRBP promoter in transgenic mice is conferred by 212 bp of the 5'-flanking region

IRBP is a photoreceptor-specific glycoprotein that has been suggested as a retinoid carrier in the visual process. Previous research has shown that 1.3 kb of 5'-flanking sequence from the human IRBP gene is sufficient to promote photoreceptor-specific expression of reporter genes in transgenic mice. To define more narrowly the sequences that promote tissue-specific expression, chimeric constructs with shorter promoters were used to generate transgenic mice. The bacterial CAT gene was fused to fragments of 706 bp or 212 bp from the 5' end of the human IRBP gene. Analysis of the three transgenic families bearing the 706 bp IRBP promoter revealed that CAT expression was confined to the neuro-retina and the pineal gland. Analysis of the four transgenic families bearing the 212 bp IRBP promoter revealed the same tissue-specific CAT expression in three families. These results establish that tissue-specific expression of IRBP can be regulated by a short 212 bp promoter which has been conserved between humans and mice.     

Highly specific expression of luciferase gene in lungs of naïve nude mice directed by prostate-specific antigen promoter

PSA promoter has been demonstrated the utility for tissue-specific toxic gene therapy in prostate cancer models. Characterization of foreign gene overexpression in normal animals elicited by PSA promoter should help evaluate therapy safety. Here we constructed an adenovirus vector (AdPSA-Luc), containing firefly luciferase gene under the control of the 5837 bp long prostate-specific antigen promoter. A charge coupled device video camera was used to non-invasively image expression of firefly luciferase in nude mice on days 3, 7, 11 after injection of 2 x 10(9)PFU of AdPSA-Luc virus via tail vein. The result showed highly specific expression of the luciferase gene in lungs of mice from day 7. The finding indicates the potential limitations of the suicide gene therapy of prostate cancer based on selectivity of PSA promoter. By contrary, it has encouraging implications for further development of vectors via PSA promoter to enable gene therapy for pulmonary diseases.     

Tissue-specific, developmental, hormonal, and dietary regulation of rat phosphoenolpyruvate carboxykinase-human growth hormone fusion genes in transgenic mice.

The cytosolic phosphoenolpyruvate carboxykinase (PEPCK) gene is expressed in multiple tissues and is regulated in a complex tissue-specific manner. To map the cis-acting DNA elements that direct this tissue-specific expression, we made transgenic mice containing truncated PEPCK-human growth hormone (hGH) fusion genes. The transgenes contained PEPCK promoter fragments with 5' endpoints at -2088, -888, -600, -402, and -207 bp, while the 3' endpoint was at +69 bp. Immunohistochemical analysis showed that the -2088 transgene was expressed in the correct cell types (hepatocytes, proximal tubular epithelium of the kidney, villar epithelium of the small intestine, epithelium of the colon, smooth muscle of the vagina and lungs, ductal epithelium of the sublingual gland, and white and brown adipocytes). Solution hybridization of hGH mRNA expressed from the transgenes indicated that white and brown fat-specific elements are located distally (-2088 to -888 bp) and that liver-, gut-, and kidney-specific elements are located proximally (-600 to +69 bp). However, elements outside of the region tested are necessary for the correct developmental pattern and level of PEPCK expression in kidney. Both the -2088 and -402 transgenes responded in a tissue-specific manner to dietary stimuli, and the -2088 transgene responded to glucocorticoid stimuli. Thus, different tissues utilize distinct cell-specific cis-acting elements to direct and regulate the PEPCK gene.