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  • Author: R. I. Richards x
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Inducible anti-sense RNA for angiotensinogen in stably transformed hepatoma cell lines

W. M. Clouston, C. J. Lloyd, and R. I. Richards

ABSTRACT

Angiotensinogen mRNA is found in many extrahepatic tissues, where it may participate in local angiotensin-generating systems. In this study we explore the feasibility of using anti-sense RNA to decrease angiotensinogen production in rat H4IIEC3 hepatoma cells. An amplifiable shuttle vector was modified to allow the production of high levels of stable anti-sense RNA from two regions of the mouse angiotensinogen gene under the control of the inducible sheep metallothionein promoter. Stably transformed, clonal cell lines expressing anti-sense RNA for angiotensinogen were isolated after selection with the aminoglycoside G418. Subsequently, the number of chromosomally integrated copies of the angiotensinogen anti-sense constructs was coamplified by methotrexate selection for dihydrofolate reductase activity carried on the shuttle vector. With a 20- to 30-fold induction of the anti-sense RNAs, the target angiotensinogen mRNA level was reduced to 25–30% of control values. The specificity of this effect was confirmed by showing no decrease in either β-tubulin or neomycin phosphotransferase mRNA levels. Using tissue-specific promoters, it should be possible to direct these effects to specific organs in transgenic mice. However, in agreement with results from other groups, our findings suggest that it will not be possible to eradicate completely the target gene product using the anti-sense RNA strategy.

DOI:
https://doi.org/10.1677/jme.0.0040107
Volume 4: Issue 2,
Pages:
107–117 (11 total)
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Differential binding of thyroid hormone receptors to mouse glandular kallikrein gene promoters: evidence for multiple binding regions in the mGK-6 gene

J. W. Barlow, L. E. Raggatt, C. C. Drinkwater, I. G. Lyons, and R. I. Richards

ABSTRACT

We have used a DNA-cellulose competition assay to investigate the binding of thyroid hormone receptors to fragments of the mouse glandular kallikrein genes and the human and rat GH genes. Nuclear extracts from human lymphoblastoid IM-9 cells were incubated with [125I]tri-iodothyronine ([125I]T3) and DNA-cellulose. The ability of cloned gene fragments to compete for radiolabelled receptors bound to DNA-cellulose was compared with that of DNA from pBR322. As previously observed, a 900 bp fragment from the human GH gene showed preferential binding to the thyroid hormone receptor. High-affinity binding was observed with a synthetic fragment of the rat GH gene encompassing positions − 163 to − 192 but not with a similar fragment from positions −224 to −192. Preferential binding was also observed with fragments of the mouse glandular kallikrein gene, mGK-6. Binding to the entire gene and fragments containing 2300 and 776 bp of the promoter region was identical. Detectable but reduced binding was seen with a shorter fragment. These results suggest that the T3 receptor binds to multiple sites within the first 776 bp of the mGK-6 gene promoter. Potential thyroid hormone response elements can be identified within this region of the gene. In contrast, the kallikrein gene mGK-3, which shows a different response to thyroid hormone from that of mGK-6, showed no significant binding in the comparable promoter region.

DOI:
https://doi.org/10.1677/jme.0.0030079
Volume 3: Issue 2,
Pages:
79–84 (6 total)
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The function of conserved elements in the promoter of the mouse angiotensinogen gene

M. Congiu, W. M. Clouston, R. T. Fernley, and R. I. Richards

ABSTRACT

The angiotensinogen gene encodes the precursor protein for the potent vasoconstrictor angiotensin II. Although the gene is expressed in several tissues, the liver is the major source of circulating protein. In previous in-vivo studies we have found that a minigene containing 750 bp of 5′-flanking sequence is transcribed in a manner which largely parallels the expression of the endogenous gene. In this report, we characterized conserved elements in the promoter region, in order to determine their role in the transcription of the angiotensinogen gene. Constructs fused to the chloramphenicol acetyl transferase (CAT) reporter gene were transfected into hepatocarcinoma Hep G2 cells as well as into non-hepatic cell lines. We found that 5′-deletion mutant constructs, containing sequences from + 25 to −90 bp and −321 to −750 bp, were each able to activate transcription. These constructs contain the TATA box and core promoter sequences, including an Spl-binding site, and two glucocorticoid responsive elements respectively. In the non-hepatic cell lines, HeLa and Jeg-3, we found that the constructs were transcribed at a much lower rate when compared with the expression of a plasmid containing the Rous sarcoma virus long terminal repeat fused to the CAT gene. Constructs which included sequence 5′ to −244 were oestrogen inducible. An element which is conserved between rodent and human angiotensinogen promoters is contained within a sequence which is oestrogen responsive, while another binds the liver-enriched transcriptional activator hepatocyte nuclear factor 1. However, the role of this transactivator in the transcription of angiotensinogen remains uncertain.

DOI:
https://doi.org/10.1677/jme.0.0090019
Volume 9: Issue 1,
Pages:
19–29 (11 total)