In vivo identification of a negative regulatory element in the mouse renin gene using direct gene transfer.

Abstract

DBA/2J mouse contains two renin gene loci (Renld and Ren2d). Ren2d but not Renl d is expressed in submandibular gland (SMG) while both are expressed in the kidney. Based on in vitro studies, we have postulated that a negative regulatory element (NRE) in the renin gene promoter is involved in its tissue-specific expression. In this study, we examined the molecular mechanism at the in vivo level using direct gene transfer. Fragments of the Renld or Ren2d promoter were fused to a chloramphenicol acetyltransferase (CAT) gene expression vector. These constructs complexed in fusogenic liposomes were injected directly into the mouse SMG or intraarterially into the mouse kidney via the renal artery. The vector containing the CAT exhibited readily detectable in vivo expressions in both SMG and kidney. In the SMG, Renld fragment containing the NRE abolished CAT expression while deletion of the NRE restored CAT expression. The homologous fragment from the Ren2d promoter did not inhibit CAT expression while deletion of the 150-bp insertion resulted in the inhibition. Cotransfection of Renld construct with Renld-NRE oligonucleotides as transcriptional factor decoy restored CAT expression. Contrary to the SMG, transfection with Renld fragment-CAT construct or Ren2d fragment-CAT construct into the kidney resulted in similar levels of CAT expression. Interestingly, human cmyc NRE oligonucleotides which share homology with Renld-NRE competed effectively with these oligonucleotides for the regulation of Renld gene expression in vivo. This NRE sequence is also homologous to silencer elements found in multiple mammalian genes, suggesting the presence of a family of NRE/NRE binding proteins regulating expression of diverse genes. (J. Clin. Invest. 1995Invest. . 96:1230Invest. -1237

Intratumoral injection of oligonucleotides to the NFκB binding site inhibits cachexia in a mouse tumor model

“…To enhance the efficiency and stability of decoy ODN further, we employed a Sendai-virus (HVJ)-liposome delivery system which is reported to bypass endocytosis, thereby avoiding degradation in lysosomes. [13][14][15] In the preliminary experiments to study the anti-cachectic effect of NF B decoy ODN, we examined the transfection of NF B decoy ODN into the tumor: (1) once a week; or (2) on 3 consecutive days a week, for 2 weeks starting 5 days after tumor inoculation. As a result, prevention of the loss of body weight, epididymal fat, gastrocnemius muscle and carcass was observed, but the difference was not significant (data not shown).…”

“…[11][12][13] Recently, a few studies have described application of the 'decoy' ODN strategy as in vivo gene therapy. 14,15 The present study provides the first evidence of in vivo application of this novel molecular approach as a therapeutic strategy for cancer cachexia.…”

“…[13][14][15] The lipid mixture (10 mg) was deposited on the sides of a flask by removal of tetrahydrofuran in a rotary evaporator. Dried lipid was hydrated in 200 l of balanced salt solution (BSS: 137 mm NaCl, 5.4 mm KCl, 10 mm Tris-HCl, pH 7.6) containing decoy ODN.…”

“…The top layer of sucrose gradient was collected for use. [13][14][15] Evaluation of anti-cachectic effect Colon26 adenocarcinoma was maintained s.c. by 10-day serial passage in BALB/c mice. In the evaluation, a fragment (2 × 2 × 2 mm) of colon26 tumor was implanted s.c. into the left flank of BALB/c mice.…”

“…Transfection of cis-element double-stranded oligodeoxynucleotides (ODN) as decoy has been reported to be a powerful new tool for the study of transcriptional regulation. [11][12][13] Transfection of double-stranded ODN corresponding to the cis sequence results in attenuation of the authentic cis-trans interaction, leading to the removal of the trans-factors from the endogenous ciselement, with subsequent modulation of gene expression. Therefore, the decoy approach enables us to treat diseases by modulation of endogenous transcriptional regulation.…”