MSCV-based retroviral plasmids expressing 3xFLAG-Sp-dCas9 for enChIP analysis
Miyuki Yuno1, Shoko Nagata1,2, Toshitsugu Fujita1,2
1Chromatin Biochemistry Research Group, Combined Program on Microbiology and Immunology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
Biology Methods & Protocols
|August 19, 2021
Summary
Engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technology was enhanced using retroviral plasmids. This improves flexibility and expands target cell options for genomic region purification and associated molecule identification.
Area of Science:
- Molecular Biology
- Genomics
- Biotechnology
Background:
- Engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) purifies specific genomic regions for associated molecule identification.
- The clustered regularly interspaced short palindromic repeats (CRISPR) system, using guide RNA (gRNA) and catalytically inactive Cas9 (dCas9), is a key enChIP component.
Purpose of the Study:
- To enhance the flexibility and expand the range of target cells for enChIP.
- To develop murine stem cell virus (MSCV)-based retroviral plasmids for expressing dCas9 for enChIP applications.
Main Methods:
- Construction of MSCV-based retroviral plasmids encoding 3xFLAG-tagged Streptococcus pyogenes dCas9 (3xFLAG-Sp-dCas9).
- Inclusion of various drug resistance genes within the retroviral plasmids for selection of transduced cells.
- Demonstration of enChIP feasibility using the developed retroviral systems.
Main Results:
- Successfully generated MSCV-based retroviral plasmids for dCas9 expression.
- Achieved purification of target genomic regions with yields comparable to existing enChIP systems.
- Validated the utility of drug resistance genes for selecting cells successfully transduced with the dCas9 constructs.
Conclusions:
- The developed retroviral plasmid system offers increased flexibility for enChIP applications.
- This system expands the choice of target cells and facilitates drug-based selection of engineered cells.
- Provides enChIP users with more options for optimizing their experimental systems.
