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Isolation of extrachromosomal elements by histone immunoprecipitation
T I Kuschak1, B C Kuschak, G M Smith
1Manitoba Institute of Cell Biology, University of Manitoba, Winnipeg, MB, Canada.
Biotechniques
|May 18, 2001
Summary
Researchers developed a new method to isolate histone-bound extrachromosomal elements (EEs), which carry active genes. This technique enables efficient purification of EEs from various cell types for further genetic analysis.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- Extrachromosomal elements (EEs) are DNA molecules outside the main chromosomes.
- Active EEs are associated with histone proteins and contain transcribing genes, such as c-myc.
- Current methods for isolating EEs can be inefficient or lack specificity.
Purpose of the Study:
- To develop a gentle, rapid, and reproducible method for isolating histone-bound EEs.
- To enable the purification of a specific population of EEs for downstream applications like cloning and analysis.
- To validate the method's effectiveness in enriching for EEs carrying specific genes.
Main Methods:
- Isolation of histone-bound EEs using immunoprecipitation targeting histone proteins.
- Utilizing 4-hydroxytamoxifen (4-HT)-activated Myc-ER-regulatable Pre-B ABM cells for EEs isolation.
- Employing fluorescent in situ hybridization of EEs (FISH-EEs) and cDNA probes (e.g., DHFR) for validation.
Main Results:
- Successfully isolated and purified histone-bound EEs from cultured cells.
- Demonstrated enrichment of EEs carrying specific genes (e.g., DHFR) after one round of immunoprecipitation.
- Confirmed the presence of actively transcribing genes within the purified EEs.
Conclusions:
- The described method provides an effective first-step purification for histone-bound EEs.
- This technique facilitates the study of EEs and their associated genes in various biological contexts.
- The method is applicable to cultured cells, primary tissues, and tumor cells, offering broad utility.