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An efficient system to establish multiple embryonic stem cell lines carrying an inducible expression unit
Shinji Masui1, Daisuke Shimosato, Yayoi Toyooka
1Laboratory for Pluripotent Cell Studies, RIKEN Center for Developmental Biology Minatojima-minamimachi 2-2-3, Kobe, Japan. masui@cdb.riken.jp
Nucleic Acids Research
|March 3, 2005
Summary
Researchers developed the ROSA-TET system for efficiently creating multiple mouse embryonic stem (ES) cell lines with tetracycline-regulated transgenes, accelerating pluripotency and differentiation studies.
Area of Science:
- Stem cell biology
- Molecular biology
- Genetics
Background:
- Mouse embryonic stem (ES) cells are crucial for studying pluripotency and differentiation.
- Regulatable transgene expression systems are vital for analyzing cellular processes.
- A method for establishing multiple ES cell lines with such systems was lacking.
Purpose of the Study:
- To develop an efficient system for creating multiple mouse ES cell lines with tetracycline-regulatable transgenes.
- To establish a robust strategy for introducing cDNAs into the ROSA26 locus for regulated expression.
Main Methods:
- Developed the ROSA-TET system utilizing a two-step process.
- Employed knock-in of a construct with loxP and mutant sequences into the ROSA26 locus.
- Utilized recombinase-mediated cassette exchange for introducing tetracycline-regulated cDNAs.
Main Results:
- The ROSA-TET system enables the efficient establishment of multiple ES cell lines.
- Both the knock-in and exchange steps demonstrated high efficiency in generating desired clones.
- The system allows for the simultaneous establishment of cell lines with various Tc-regulated cDNAs.
Conclusions:
- The ROSA-TET system provides an effective strategy for generating multiple ES cell lines with tetracycline-regulatable transgenes.
- This system can be readily applied to various ES cell lines.
- The ROSA-TET system is expected to accelerate molecular biology research using ES cells.