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  • Alternative splicing Kidney Laminins Whole-mount in situ hybridization Organ culture Mouse  (1)
  • Basement membrane  (1)
  • 1
    ISSN: 1434-0879
    Keywords: Key words Kidney perfusion ; Glomeruli ; Gene transfer ; Alport syndrome ; Basement membrane
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract In an attempt to develop gene therapy for Alport syndrome, we have evaluated surgical methods for gene transfer into pig kidneys. For gene transfer we used an adenovirus expressing the Escherichia coliβ-galactosidase gene as a reporter gene. The viral preparation was first infused in vivo into the porcine renal artery. Then explanted kidneys were perfused ex vivo at body temperature for 12 hours with the viral solution and, finally the kidney perfusions were carried out in vivo via laparotomy for 60 and 120 minutes. Gene transfer was determined visually on histological cryosections after 5-bromo-4-chloro-3-indoyl-β-galactopyranoside (X-gal) and periodic acid-Schiff (PAS) staining. Perfusion of whole porcine kidneys ex vivo resulted in strong expression in about 80% of glomeruli. The in vivo kidney perfusion via laparotomy for 120 minutes resulted in reporter gene expression of about 75% of the glomeruli examined after 4 days. Expression was observed almost exclusively in glomeruli, while little if any expression was found in other renal structures. The present results suggest that operatively performed kidney perfusion may be used for gene transfer in treatment of glomerular disease. This surgical approach may also prove useful for somatic gene therapy of other organs.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1432-0878
    Keywords: Alternative splicing Kidney Laminins Whole-mount in situ hybridization Organ culture Mouse
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. Laminins are large heterotrimeric basement membrane proteins that consist of α, β, and γ chains. We have previously shown that the human γ2 and γ2* transcripts result from the alternative use of the LAMC2 gene 3'-end exons. To explore the biological significance of the alternative γ2 transcripts, we isolated the cDNA coding for the mouse laminin γ2* transcript, characterized the 3'-end of the murine LAMC2 gene, and studied the expression of alternative γ2 transcripts in several mouse tissues. The sequence reported here is the first one containing a full-length γ2* 3'-UTR from any species. The mouse γ2* transcript is 4110 bases and encodes a putative polypeptide of 1110 amino acids. This polypeptide lacks the C-terminal cysteine residue thought to be important for heterotrimer formation. The mouse γ2* transcript was found to be expressed in several tissues by polymerase chain reaction (PCR), but at very low levels. The clearest signals were obtained on embryonic day 7, and in heart and testis of adult tissues. When the laminin γ2* transcript expression pattern was compared with that of the γ2 chain, a similar tissue distribution was observed. There was, however, a significant difference in expression levels. The longer γ2 transcript was found to be much more abundant than the shorter γ2* variant. Moreover, by whole-mount in situ hybridization, the shorter γ2* form was localized in the mesenchyme of the developing kidney whereas the longer γ2 form was exclusively present in the epithelium of the Wolffian (nephric) duct and ureteric bud. The results indicate different functions for the γ2 variants.
    Type of Medium: Electronic Resource
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