Tissue-specific expression in transgenic mice directed by the 5'-flanking sequences of the human gene encoding interphotoreceptor retinoid-binding protein

Interphotoreceptor retinoid-binding protein (IRBP) is an extracellular protein that has been suggested to participate in the visual process as a carrier for visual retinoids. A chimeric gene composed of the human IRBP promoter fused to the bacterial reporter gene chloramphenicol acetyltransferase (CAT) was used to generate transgenic mice. Analysis of six transgenic families revealed that the CAT gene, concomitant with the endogenous IRBP gene, was expressed primarily in the retina and, to a lesser extent, in the pineal gland. These results establish that a 1.3-kilobase fragment from the 5' end of the human IRBP gene is sufficient to direct transgene expression to a visual subdivision of the central nervous system.     

Functional dissection of the promoter of the interphotoreceptor retinoid-binding protein gene: the cone-rod-homeobox element is essential for photoreceptor-specific expression in vivo.

The essential control elements in the interphotoreceptor retinoid-binding protein gene (IRBP) promoter are located between -156 and +19. The -156/-109 sequence contains a retina-specific DNAse I footprint and shows a positive regulatory activity in transiently transfected retinoblastoma cells. The -105/-85 sequence is G/C rich, shows a non-tissue specific DNAse I hypersensitivity, and a negative regulatory activity in retinoblastoma cells. The -76/-42 sequence shows a retinal-specific footprint and contains a "cone-rod-homeobox element" (CRXE) and a "photoreceptor conserved element" (PCE). IRBP promoter fragments with mutations in either CRXE, PCE or in both were linked to reporter genes and analyzed both by transient transfection and in transgenic mice. In retinoblastoma cells, the mutated CRXE-containing promoter shows a 60% repression of the CAT activity whereas the mutated PCE-containing promoter shows a 30% repression. In HeLa cells transfected with these promoters, co-transfection of a Crx expression vector with wild-type, but not with CRXE mutant promoter, activates CAT activity 20-fold over the background activity. Mutation of PCE alone or conversion of CRXE to PCE reduces this Crx-activated CAT activity to only 4-fold over the background activity. In the transgenic mouse experiments, none of the 12 lines with CRXE mutant promoter show significant expression of lacZ in the retina. In contrast, 9 of the 17 transgenic lines with PCE mutant promoter show photoreceptor-specific lacZ expression. Thus the Crx interaction with CRXE is essential for the photoreceptor-specific activity of the IRBP promoter in vivo. This interaction does not appear to require PCE, but is enhanced when PCE is present.     

Analysis of the Alternative Promoters that Regulate Tissue-Specific Expression of Human Aromatic l-Amino Acid Decarboxylase

Abstract: Previously we identified two alternative first exons (exon N1 and exon L1) coding for 5' untranslated regions of human aromatic l -amino acid decarboxylase (AADC) and found that their alternative usage produced two types of mRNAs in a tissue-specific manner. To determine the cis -acting element regulating the tissue-specific expression of human AADC, we produced three kinds of transgenic mice harboring 5' flanking regions of the human AADC gene fused to the bacterial chloramphenicol acetyltransferase (CAT) gene. The transgene termed ACA contained −7.0 kb to −30 bp in exon N1, including the entire exon L1; ACN contained −3.6 kb to −30 bp in exon N1; and ACL contained −2.8 kb to −42 bp in exon L1. The ACA transgenic mice expressed CAT at extremely high levels in peripheral nonneuronal tissues, such as pancreas, liver, kidney, small intestine, and colon, that contained endogenous high AADC activity, whereas CAT immunoreactivity was not detected in either catecholaminergic or serotonergic neurons in the CNS. Thus, it was suggested that the ACA transgene contained the major part of cis -regulatory elements for the expression of AADC in peripheral nonneuronal tissues. On the other hand, the ACN transgenic mice moderately expressed CAT in various tissues except for the lung and liver, and the ACL transgenic mice showed moderate CAT expression only in the kidney.     

Activity of the adenosine deaminase promoter in transgenic mice.

The promoter of the human gene for adenosine deaminase (ADA) is extremely G/C-rich, contains several G/C-box motifs (GGGCGGG) and lacks any apparent TATA or CAAT boxes. These features are commonly found in promoters of genes that lack a strong tissue specificity, and are referred to as "housekeeping genes". Like other housekeeping genes, the ADA gene is expressed in all tissues. However, there is a considerable variation in the levels of expression of the ADA protein in different tissues. In order to study the activity of the ADA promoter, transgenic mice were generated that harbor a chimeric gene composed of the ADA promoter linked to a reporter gene encoding the bacterial enzyme Chloramphenicol Acetyl Transferase (CAT). These mice reproducibly showed CAT expression in all tissues examined, including the hemopoietic organs (spleen, thymus and bone marrow). However, examination of the actual cell types expressing the CAT gene revealed the ADA promoter to be inactive in the hemopoietic cells. This was substantiated by a transplantation experiment in which bone marrow from ADA-CAT transgenic mice was used to reconstitute the hemopoietic compartment of lethally irradiated mice. The engrafted recipients revealed strongly reduced CAT activity in their hemopoietic organs. The lack of expression in hemopoietic cells was further shown to be correlated with a hypermethylated state of the transgene. Combined, our data suggest that the ADA promoter sequences tested can direct expression in a wide variety of tissues as expected for a regular housekeeping gene promoter. However, the activity of the ADA promoter fragment did not reflect the tissue-specific variations in expression levels of the endogenous ADA gene. Additionally, regulatory elements are needed for expression in the hemopoietic cells.     

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.     

Hormonal and nutritional regulation of muscle carnitine palmitoyltransferase I gene expression in vivo

Transgenic mice carrying the human heart muscle carnitine palmitoyltransferase I (M-CPTI) gene fused to a CAT reporter gene were generated to study the regulation of M-CPTI gene expression. When the mice were fasted for 48 h, CAT activity and mRNA levels increased by more than 2-fold in heart and skeletal muscle, but not liver or kidney. In the diabetic transgenic mice, there was a 2- to 3-fold increase in CAT activity and CAT mRNA levels in heart and skeletal muscle which upon insulin administration reverted to that observed with the control insulin sufficient transgenic mice. Feeding a high fat diet increased CAT activity and mRNA levels by 2- to 4-fold in heart and skeletal muscle of the transgenic mice compared to the control transgenic mice on regular diet. Overall, the M-CPTI promoter was found to be necessary for the tissue-specific hormonal and dietary regulation of the gene expression.     

Development of photoreceptor-specific promoters and their utility to investigate EIAV lentiviral vector mediated gene transfer to photoreceptors

BACKGROUND: We wanted to investigate the ability of recombinant equine infectious anemia virus (EIAV) vectors to transduce photoreceptor cells by developing a series of photoreceptor-specific promoters that drive strong gene expression in photoreceptor cells. METHODS: Promoter fragments derived from the rhodopsin (RHO), the beta phosphodiesterase (PDE) and the retinitis pigmentosa (RP1) genes were cloned in combination with an enhancer element, derived from the interphotoreceptor retinoid-binding protein gene (IRBP), into luciferase reporter plasmids. An in vitro transient reporter assay was carried out in the human Y-79 retinoblastoma cell line. The optimal promoters from this screen were then cloned into the recombinant EIAV vector for evaluation in vivo following subretinal delivery into mice. RESULTS: All promoters maintained a photoreceptor-specific expression profile in vitro and the gene expression was further enhanced in combination with the IRBP enhancer. The use of IRBP-combined RHO or PDE promoters showed modest but exclusive expression in photoreceptors following subretinal delivery to mice. By contrast an EIAV vector containing the cytomegalovirus (CMV) promoter drove reporter gene expression in both photoreceptors and retinal pigment epithelium. CONCLUSIONS: It may be possible to use recombinant EIAV vectors containing photoreceptor-specific promoters to drive therapeutic gene expression to treat a range of retinal degenerative diseases where the photoreceptor cell is the primary disease target.