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Development of a multiple-gene-loading method by combining multi-integration system-equipped mouse artificial
Kazuhisa Honma1, Satoshi Abe2, Takeshi Endo3
1Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Graduate School of Medical Science, Tottori University, Yonago, Tottori, Japan.
Plos One
|March 6, 2018
Summary
Researchers developed a new method using CRISPR-Cas9 to enable mouse artificial chromosome (MAC) vectors to carry multiple gene-loading vectors (GLVs). This advance overcomes drug resistance limitations for advanced gene delivery applications.
Area of Science:
- Genetics and Genomics
- Molecular Biology
- Biotechnology
Background:
- Mouse artificial chromosomes (MACs) offer advantages for gene delivery, including stable maintenance and large DNA capacity.
- A multi-integrase MAC (MI-MAC) platform was designed for transferring multiple genes but faced limitations in selecting integrated gene-loading vectors (GLVs).
- Limited drug resistance genes (DRGs) hinder the selection of multiple GLVs within the MI-MAC system.
Purpose of the Study:
- To develop novel methods for integrating multiple GLVs into the MI-MAC.
- To overcome the limitations of selecting multiple GLVs by reusing drug resistance genes (DRGs) via CRISPR-Cas9.
- To demonstrate the successful integration and expression of five GLVs within the MI-MAC.
Main Methods:
- Utilized the CRISPR-Cas9 system to knock out and enable the reuse of drug resistance genes (DRGs).
- Developed new methodologies for the integration of multiple gene-loading vectors (GLVs) into the mouse artificial chromosome (MAC) vector.
- Introduced five GLVs, each encoding a distinct fluorescent or luminescent protein (EGFP, mCherry, BFP, Eluc, Cluc), into the MI-MAC.
Main Results:
- Successfully integrated five GLVs, each encoding a fluorescent or luminescent reporter, into the MI-MAC using the developed CRISPR-Cas9 based methods.
- Demonstrated stable and functional expression of genes of interest (GOI) from the MI-MAC without silencing in host cells.
- Confirmed successful transfer of the MI-MAC carrying five GLVs to recipient cells via microcell-mediated chromosome transfer (MMCT), with all five signals detected.
Conclusions:
- The MI-MAC, enhanced with CRISPR-Cas9 technology for DRG reuse, effectively functions as a multiple-GLV integration vector.
- This approach overcomes previous bottlenecks in selecting multiple integrated GLVs, enabling more complex gene delivery.
- The developed MI-MAC system holds significant potential for creating advanced multiple-gene models, including humanized, monitoring, and disease models, as well as for reprogramming and inducible gene expression.
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