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Cre-mediated site-specific cassette exchange in erythroid cell
Xing-Guo Li1, Hao-Heng Yan, De-Pei Liu
1National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences Peking Union Medical College, Beijing 100005, China.
Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao Acta Biochimica Et Biophysica Sinica
|September 30, 2003
Summary
Cre-mediated cassette exchange enabled site-specific integration of DNA elements in mouse erythroleukemia cells. Investigating the human beta-globin HS2 element revealed orientation-dependent gene silencing, impacting EGFP expression.
Area of Science:
- Genetics
- Molecular Biology
- Cell Biology
Background:
- Cre-mediated cassette exchange is a powerful tool for site-specific integration of DNA sequences into the genome.
- Understanding the influence of chromatin context and DNA element orientation on gene expression is crucial for genetic engineering and gene therapy applications.
Purpose of the Study:
- To investigate the function of the erythroid cis-acting DNA element, specifically the human beta-globin gene cluster 5' DNase I hypersensitive site 2 (HS2).
- To examine the impact of site-specific integration and orientation on gene expression within specific chromatin contexts using mouse erythroleukemia cells.
Main Methods:
- Cre-mediated cassette exchange with inverted Lox sites for site-specific integration.
- Selection of hygromycin-resistant clones and confirmation of single-copy integration via PCR and Southern blotting.
- Analysis of EGFP (Enhanced Green Fluorescent Protein) gene expression using flow cytometry after cassette exchange.
Main Results:
- Single-copy integration of the target vector was confirmed in selected clones.
- A 732-bp fragment of human beta-globin HS2 was successfully integrated into clone A in an anti-genomic orientation.
- Low EGFP expression was observed in the clone with HS2 integration, suggesting orientation-dependent gene silencing.
Conclusions:
- Site-specific integration using Cre-mediated cassette exchange is effective for introducing DNA elements into specific chromosomal locations.
- The orientation of the integrated HS2 element significantly influences gene expression, potentially leading to gene silencing in non-permissive orientations.
- These findings highlight the importance of orientation and chromatin context in the design of gene expression strategies.