Lentiviral-mediated RNAi in vivo silencing of Col6a1, a gene with complex tissue specific expression pattern.
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.
Frka
Kosjenka
K
Department of Histology Microbiology and Medical Biotechnology, University of Padova, Padova, Italy.
Facchinello
Nicola
N
Del Vecchio
Claudia
C
Carpi
Andrea
A
Curtarello
Matteo
M
Venerando
Rina
R
Angelin
Alessia
A
Parolin
Cristina
C
Bernardi
Paolo
P
Bonaldo
Paolo
P
Volpin
Dino
D
Braghetta
Paola
P
Bressan
Giorgio M
GM
D000818:Animals; D017209:Apoptosis; D015152:Blotting, Northern; D002460:Cell Line; D024142:Collagen Type VI / Q000235:genetics / Q000378:metabolism*; D005260:Female; D020869:Gene Expression Profiling; D005822:Genetic Vectors / Q000235:genetics; D015151:Immunoblotting; D016086:Lentivirus / Q000235:genetics*; D008297:Male; D053078:Membrane Potential, Mitochondrial; D051379:Mice; D008822:Mice, Transgenic; D041681:NIH 3T3 Cells; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction
C540085:Col6a1 protein, mouse; D024142:Collagen Type VI