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Functional cloning of drug resistance genes from retroviral cDNA libraries
Peter T Bosma1, Simone J van Eert, Nicolaas G J Jaspers
1Department of Medical Oncology, Josephine Nefkens Institute, 3000 DR Rotterdam, The Netherlands.
Abstract:
To improve the curative success of chemotherapy, it will be essential to understand the molecular basis of drug resistance (DR) and sensitivity. We have developed a cell culture system that enables the functional cloning of mammalian DR genes based on phenotypic selection after overexpression of mammalian retroviral cDNA libraries and validated our system using the anticancer drug cisplatin. ERCC1-deficient and therefore cisplatin-hypersensitive mouse embryonic fibroblast target cells were transduced with a human placenta retroviral cDNA library. Subsequent cisplatin selection yielded 20 DR clones, each containing a recurring human ERCC1 gene. Surprisingly, nine of these clones contained 5'-truncated ERCC1 sequences that required alternative splicing of the vector sequence to encode a functional ERCC1 protein. The usage of cryptic splice sites in the vector sequence should be taken into consideration when interpreting results from retroviral gene expression applications, and might have consequences for the safe application of retroviral constructs in gene therapy.
Insights
Understanding drug resistance (DR) is key to improving chemotherapy. This study developed a cell system to clone DR genes, identifying ERCC1 as crucial for cisplatin resistance and revealing unexpected alternative splicing in retroviral applications.
Area of Science:
- Molecular Biology
- Genetics
- Cancer Research
Background:
- Improving chemotherapy efficacy requires understanding drug resistance (DR) and sensitivity at a molecular level.
- Mammalian retroviral cDNA libraries can be used to identify genes involved in DR.
Purpose of the Study:
- To develop and validate a cell culture system for functional cloning of mammalian DR genes.
- To identify genes conferring resistance to the anticancer drug cisplatin.
Main Methods:
- Overexpression of a human placenta retroviral cDNA library in ERCC1-deficient mouse embryonic fibroblast cells.
- Phenotypic selection using cisplatin to isolate drug-resistant clones.
- Sequencing of DR clones to identify the conferring genes.
Main Results:
- A functional cloning system for mammalian DR genes was successfully developed and validated.
- Twenty cisplatin-resistant clones were isolated, all containing the human ERCC1 gene.
- Nine clones contained truncated ERCC1 sequences that required alternative splicing of vector sequences for functional protein expression.
Conclusions:
- The ERCC1 gene is critical for conferring resistance to cisplatin.
- Alternative splicing, including the use of cryptic splice sites in retroviral vectors, can occur and must be considered when interpreting results.
- Findings have implications for the safe application of retroviral constructs in gene therapy.