Characteristics Episomal gene expression; therefore little risk of insertional mutagenesis. Infects dividing and non-dividing cells. Transient high-level protein expression. Lower host immune response due to no viral proteins being expressed by the helper dependent virions. Disadvantages and Adverse Effects The viral particles themselves may cause an immune response, but it is typically less intense than a first generation adenovirus.
Viral particles can be neutralized by the host immune response. Transient expression of the transgene due to lack of integration into the host genome.
Low level helper virus contamination is present in every preparation. The helper virus is a first generation adenovirus that does not express any transgenes, but does express viral proteins that may be toxic. The helper virus genome retains the ability to replicate and provide all the functions necessary in trans for the replication and packaging of a vector lacking most Ad sequences Fig.
Generation of Ad-based vectors complemented by a helper virus containing a lox P-flanked packaging signal. Infection of Cre-expressing cells with the helper virus results in excision of the viral packaging signal, rendering the helper virus DNA unpackagable. The helper virus will then provide all of the functions necessary in trans for replication and packaging of an Ad vector containing appropriate cis-acting elements [i.
Since all functions necessary for virion formation are provided by the helper virus, the majority of the Ad sequences contained in the vector can be replaced by a foreign gene and other non-Ad sequences stuffer. The titer of the vector can be increased by serial passage in helper virus-infected Cre cells.
Cell growth, virus propagation, and titration was carried out as previously described 1. The derived cell lines that stably express the Cre recombinase, Cre1 and Cre4 L. Plasmids were constructed according to standard protocols The AdLC8 helper virus Fig. A synthetic lox P site with Bam HI compatible ends, obtained by annealing two single-stranded oligonucleotides 32 , was cloned into pLC4 at a unique Bam HI site, located at nucleotide from the left end of the Ad5 genome, generating pLC5.
Previously, this region was shown to tolerate similar DNA insertions without affecting virus replication Thus, the resulting plasmid, pLC8, contains two lox P sites, separated by bp, and flanking the Ad packaging signal and neo cassette. Details of the construction of AdLC8cluc are available on request. A AdLC8 contains two lox P sites flanking the viral packaging signal and a neomycin gene expanded , and was constructed as outlined in the Materials and Methods.
The E1 and E3 deletions in AdLC8 remove sequences located between bp and 28,—30, bp of the Ad genome, respectively. A Pvu I restriction map of AdLC8, with fragment sizes indicated in kb, is shown above the schematic linear map, and Pvu I restriction sites in the expanded regions are indicated P.
Cre-mediated recombination between the two lox P sites results in the disappearance of the 1. B or Cre1 cells were infected with AdLC8 at a m. The 9. C DNA from the agarose gel shown in B was transferred to a nylon membrane and hybridized with a probe derived from pLC8 to monitor early Cre-mediated excision events. The 0—9-hr lanes were exposed to x-ray film for 1 hr, and the 12—hr lanes for 10 sec.
Fragments of unrecombined AdLC8 are indicated by open arrows and recombination products by filled arrows. Molecular sizes kb , are given on the left and right margins. Thus, pUMA10R retains Ad-specific sequences corresponding to bp of the left end and bp of the right end of Ad5. The cells were scraped into the medium and the virus released by three rounds of freezing and thawing.
The infected monolayers were washed once with PBS, fixed with 0. Plates were incubated overnight at room temperature, and bfu determined. Viral lysates were also monitored for helper virus and RCA by plaque assays on and A monolayers, respectively. Large-scale virus preparations of AdRP were prepared by infecting mm dishes of Cre with 1 ml of crude AdRP stock per mm dish m.
After complete cytopathic effect, purification of AdRP virions was performed by CsCl buoyant density centrifugation as described 1 , and fractions were collected through the viral bands, and assayed for luciferase-expressing virus after dilution and infection of cells.
Crude protein extracts were prepared 24 hr postinfection, and assayed for luciferase activity using a commercial kit Promega and a Berthold model LB luminometer. The quantity of luciferase present in each sample was calculated by comparison with a standard curve of luminescence vs. The Cre recombinase, when expressed in cells either transiently from an Ad vector 32 or stably from an integrated copy of the gene L.
Cloning lox P sites flanking the Ad packaging signal would limit the packaging of a helper virus when Cre is expressed, while allowing it to produce all of the proteins necessary in trans to replicate and package a second vector Fig. Since the cis-acting elements required for viral DNA replication are independent of those required for packaging of viral DNA into virions, removal of the packaging signal should not impair viral DNA replication, allowing the synthesis of sufficient template for expression of late viral genes to provide helper functions.
We first constructed the helper virus AdLC8 Fig. DNA was purified at various times postinfection, digested with Pvu I, separated on a 0. Instead, a 3. The excision product of 1. Furthermore, Fig. To further examine the kinetics of Cre-mediated recombination, Southern blot analysis was performed. DNA from the agarose gel shown in Fig. The probe hybridized with fragments of , , , , and bp of unrecombined AdLC8 Fig.
Overexposure of the membrane during autoradiography revealed that no excision occurred prior to 6 hr postinfection in the Cre1 line, suggesting that the extreme left end of the virus may not be accessible to Cre during early times after infection, even though Cre is present in Cre1 cells at the time of infection. The excised circle represented by a 1.
No recombination products 3. Since the quantity of unrecombined viral DNA increased with kinetics similar to that of the total viral DNA, we conclude that the unrecombined fraction is from actively replicating virus.
It is not known at present why these viral genomes escape Cre-mediated recombination. Nevertheless, the packaging signal was efficiently excised from the majority of the AdLC8 virus in the Cre1 cell line, and removal of this sequence did not affect viral DNA replication.
Removal of the packaging signal from AdLC8 in Cre1 cells should allow AdLC8 to provide helper-functions for Ad vectors that lack most, if not all, Ad coding sequences. RCA were detected after the second amplification through Cre4, and RCA titers continued to increase with subsequent passages, accounting for the increase in infectious virus after passage 5.
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