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Construction and characterization of a replication-competent retroviral shuttle vector plasmid
Jangsuk Oh1, John G Julias, Andrea L Ferris
1HIV Drug Resistance Program, National Cancer Institute at Frederick, National Institutes of Health, Frederick, Maryland 21702-1201, USA.
Journal of Virology
|January 19, 2002
Summary
We developed RSVP vectors for cloning viral DNA. These vectors facilitate the rescue of both unintegrated and integrated viral DNA from avian cells into bacteria for further study.
Area of Science:
- Molecular Biology
- Virology
- Biotechnology
Background:
- Retroviral vectors are crucial tools in molecular biology and virology.
- Efficient methods for cloning and manipulating viral DNA are essential for research.
Purpose of the Study:
- To develop novel retroviral shuttle vectors (RSVP) for efficient cloning of avian retroviral DNA.
- To enable the rescue and recovery of both unintegrated and integrated proviral DNA from avian cells.
Main Methods:
- Construction of two RSVP vector variants containing zeocin or blasticidin resistance genes.
- Transfection of RSVP vectors into avian DF-1 cells.
- Rescue of viral DNA from transfected cells using bacterial transformation and lac repressor enrichment.
- Analysis of vector stability using Southern blot.
Main Results:
- RSVP vectors successfully enabled the rescue of both circularized unintegrated and integrated proviral DNA into *Escherichia coli*.
- Lac repressor-mediated enrichment significantly aided in the recovery of vector DNA.
- Electroporation yielded higher rescue efficiency compared to standard transformation.
- RSVP vectors demonstrated stability for approximately three viral passages in avian cells.
Conclusions:
- RSVP vectors are effective tools for cloning unintegrated and integrated retroviral DNAs.
- The developed method simplifies the recovery and manipulation of viral DNA for further research.
- RSVP vectors offer a valuable platform for genetic studies involving avian retroviruses.