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Psi- vectors: murine leukemia virus-based self-inactivating and self-activating retroviral vectors
K A Delviks1, W S Hu, V K Pathak
1Department of Genetics and Developmental Biology, and Mary Babb Randolph Cancer Center, West Virginia University, Morgantown 26506, USA.
Abstract:
We have developed murine leukemia virus (MLV)-based self-inactivating and self-activating vectors to show that the previously demonstrated high-frequency direct repeat deletions are not unique to spleen necrosis virus (SNV) or the neomycin drug resistance gene. Retroviral vectors pKD-HTTK and pKD-HTpTK containing direct repeats composed of segments of the herpes simplex virus type 1 thymidine kinase (HTK) gene were constructed; in pKD-HTpTK, the direct repeat flanked the MLV packaging signal. The generation of hypoxanthine-aminopterin-thymidine-resistant colonies after one cycle of retroviral replication demonstrated functional reconstitution of the HTK gene. Quantitative Southern analysis indicated that direct repeat deletions occurred in 57 and 91% of the KD-HTTK and KD-HTpTK proviruses, respectively. These results demonstrate that (i) deletion of direct repeats occurs at similar high frequencies in SNV and MLV vectors, (ii) MLV psi can be efficiently deleted by using direct repeats, (iii) suicide genes can be functionally reconstituted during reverse transcription, and (iv) the psi region may be a hot spot for reverse transcriptase template switching events.
Insights
High-frequency direct repeat deletions are not unique to spleen necrosis virus (SNV) vectors. Murine leukemia virus (MLV) vectors also show these deletions, demonstrating MLV psi can be deleted and suicide genes can be reconstituted.
Area of Science:
- Molecular Biology
- Virology
- Gene Therapy
Background:
- Direct repeat sequences are known to cause deletions in retroviral vectors.
- Previous studies focused on spleen necrosis virus (SNV) and neomycin resistance genes.
Purpose of the Study:
- To investigate if high-frequency direct repeat deletions are unique to SNV vectors.
- To assess direct repeat deletions in murine leukemia virus (MLV)-based vectors.
- To determine if MLV packaging signal (psi) can be deleted by direct repeats.
Main Methods:
- Construction of MLV-based retroviral vectors (pKD-HTTK and pKD-HTpTK) containing herpes simplex virus type 1 thymidine kinase (HTK) gene segments as direct repeats.
- Generation of hypoxanthine-aminopterin-thymidine-resistant colonies to assess functional gene reconstitution.
- Quantitative Southern analysis to determine the frequency of direct repeat deletions.
Main Results:
- Functional reconstitution of the HTK gene was demonstrated after one cycle of retroviral replication.
- Direct repeat deletions occurred in 57% of KD-HTTK and 91% of KD-HTpTK proviruses.
- High-frequency deletions were observed in both SNV and MLV vectors.
Conclusions:
- Direct repeat deletions occur at similar high frequencies in both SNV and MLV vectors.
- MLV psi can be efficiently deleted using direct repeats.
- Suicide genes can be functionally reconstituted during reverse transcription, and the psi region may be a hot spot for template switching.