Using RT-prone recombination to promote re-building of complete retroviral vectors from two defective precursors: low efficiency and sequence specificities.
Retroviral recombination has been suggested as a useful way to modify retroviral vectors. The possibility to combine two multiply deleted retroviral vectors into a novel vector was evaluated. To investigate this possibility we have constructed two defective vectors containing a shared internal ribosome entry site (IRES). The IRES was selected for its complex secondary structure, a feature described to favour retroviral recombination. The IRES was expected to promote a recombination event leading to the formation of a unique, functional retroviral vector. By supporting expression of two transgenes from a single promoter, this sequence was also expected to allow straightforward detection of the recombination event. The present data confirms the achievement of recombination-dependent rescue, albeit at low efficiency. Unexpectedly, a preferential use of the packaging signal (Psi) for recombination was observed, as compared to the IRES. Together these observations mitigate the idea of using this technique for the design of retroviral vectors.
Bru
Thierry
T
Université François Rabelais Tours, INSERM ERI 19, IFR 136, Faculté de Médecine, 10 Bd Tonnellé, France.
Galetto
Román
R
Piver
Eric
E
Collin
Christine
C
Negroni
Matteo
M
Pagès
Jean-Christophe
JC
D002460:Cell Line; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D011995:Recombination, Genetic; D012190:Retroviridae / Q000235:genetics*; D048348:Reverse Transcription; D012270:Ribosomes / Q000378:metabolism; D014161:Transduction, Genetic; D014162:Transfection; D019076:Transgenes / Q000235:genetics / Q000528:radiation effects; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D049452:Green Fluorescent Proteins; D001616:beta-Galactosidase