(NAS Colloquium) Genetic Engineering of Viruses and Viral Vectors (1996) / Chapter Skim
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Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector
Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector
Pages 11382-11388
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From page 11382... ...
Box 85800, San Diego, CA 92186-5800 ABSTRACT We describe the construction of a safe, replication-defective and efficient lentiviral vector suitable for in vivo gene delivery. The reverse transcription of the vector was -- -I found to be a rate-limiting step; therefore, promoting the reaction inside the vector particles before delivery signifi cantly enhanced the efficiency of gene transfer.
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From page 11383... ...
For immunofluorescence labeling, mouse monoclonal anti-NeuN and guinea pig anti-GFAP antibodies, secondary antibodies coupled to the fluorescent markers CY5, dichlorotriazinyl amino fluorescein, and Texas Red were also used, and the mounted sections were analyzed by confocal scanning laser microscopy (Bio-Rad model MRTC600~. Fluorescent signals were collected, digitally color enhanced, and superimposed.
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From page 11384... ...
As the transfectant conditioned medium contained, on average, 4 x 105 transducing units/ml, titers of 2-4 x 108 transducing units/ml were routinely achieved. When normalized to the content of p24 Gag antigen, the transducing activity of the vector was not affected by the centrifugation steps, averaging 4500 transducing units per ng of p24 in 208F fibroblasts.
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From page 11385... ...
Colloquium Paper: Naldini et al. 2 weeks HIV dNTP's' MLV dNTP's+ HIV Integrase Mutant dNTP's' 3 months HIV dNTP's~ MLV dNTP's' Proc.
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From page 11386... ...
. While the mutation severely decreases the activity of the enzyme in vitro and in viva, it has no detectable effect on the preceding steps of the infection pathway, including particle budding, entry into the target cell, reverse transcription, and nuclear import.
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From page 11387... ...
The novel feature of the packaging plasmid described in this paper precludes the generation of wild-type HIV viruses, even by unlikely rearrangement and recombination events, given the actual absence of most of HIV env sequences in all three plasmids. In the previously described plasmid pCMV7\R9, the env reading frame was blocked by insertion of a linker containing multiple stop codons.
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From page 11388... ...
11388 Colloquium Paper: Naldini et al.
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