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. 1989 May;86(9):3065–3069. doi: 10.1073/pnas.86.9.3065

Integration of mini-retroviral DNA: a cell-free reaction for biochemical analysis of retroviral integration.

T Fujiwara 1, R Craigie 1
PMCID: PMC287065  PMID: 2524066

Abstract

After retroviral infection of a permissive cell, the viral RNA is reverse-transcribed to make a DNA copy of the viral genome. Integration of this DNA copy into the host genome is a necessary step for efficient viral replication. We have developed a cell-free system for integration of exogenous mini-retroviral DNA. The termini of this linear mini-Moloney murine leukemia virus (MoMLV) DNA are designed to mimic the ends of authentic unintegrated MoMLV DNA. The viral proteins required for integration can be provided either as a cytoplasmic extract of MoMLV-infected NIH 3T3 cells or as disrupted MoMLV particles. Phage lambda DNA serves as the target for integration. Genetic markers present on the mini-MoMLV DNA enable integration events to be detected, and the recombinants recovered, by selection in Escherichia coli. Integration, which occurs at heterogeneous locations in the target DNA, is absolutely dependent on the presence of a source of viral proteins and a divalent cation in the reaction mixture. The fidelity of the integration reaction was confirmed by sequencing the junctions between the integrated MoMLV DNA and adjacent lambda DNA sequence. In each case, as expected for authentic MoMLV DNA integration, a 4-base-pair duplication of target DNA sequence flanked the integrated MoMLV DNA.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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