Lentiviral-mediated RNAi in vivo silencing of Col6a1, a gene with complex tissue specific expression pattern

Kosjenka Frka, Nicola Facchinello, Claudia Del Vecchio, Andrea Carpi, Matteo Curtarello, Rina Venerando, Alessia Angelin, Cristina Parolin, Paolo Bernardi, Paolo Bonaldo, Dino Volpin, Paola Braghetta, Giorgio M. Bressan

Research output: Contribution to journalArticlepeer-review

Abstract

RNA interference (RNAi) through the use of lentiviral vectors is a valuable technique to induce loss of function mutations in mammals. Although very promising, the method has found only limited application and its general applicability remains to be established. Here we analyze how different factors influence RNAi mediated silencing of Col6a1, a gene of the extracellular matrix with a complex pattern of tissue specific expression. Our results, obtained with vectors pLVTHM and pLVPT-rtTRKRAB, point out three parameters as major determinants of the efficiency of interference: the choice of interfering sequence, the number of proviral copies integrated into the mouse genome and the site of insertion of the provirus. Although low copy number may produce efficient interference with low frequency, the general trend is that the number of integrated proviral copies determines the level of silencing and the severity of phenotypic traits. The site of insertion not only determines the overall intensity of expression of the small interfering RNA (siRNA), but also introduces slight variability of silencing in different organs. A lentiviral vector (pLVPT-rtTRKRAB) with doxycycline-inducible production of siRNA was also tested. Control of expression by the drug was stringent in many tissues; however, in some tissues turning off of siRNA synthesis was not complete. The data support the application of lentiviral vectors used here in transgenesis.

Original languageEnglish
Pages (from-to)8-17
Number of pages10
JournalJournal of Biotechnology
Volume141
Issue number1-2
DOIs
Publication statusPublished - Apr 20 2009

Keywords

  • Collagen VI
  • Lentiviral vectors
  • RNA interference
  • Transgenic mice

ASJC Scopus subject areas

  • Biotechnology
  • Applied Microbiology and Biotechnology

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