Overview
The Rosa26 locus (Gt(ROSA)26Sor) is a ubiquitously expressed safe harbor site on mouse chromosome 6 that has become the standard integration site for targeted transgene expression. Rosa26 supports stable, position-independent expression without disrupting neighboring genes or causing insertional mutagenesis, making it ideal for constitutive transgenes, conditional expression systems, Cre driver lines, and reporter alleles.
Why Rosa26 is a Safe Harbor
Rosa26 was identified as a gene trap integration site with ubiquitous expression and no discernible phenotype when disrupted. The locus is transcriptionally active in all tissues, preventing transgene silencing. Integration at Rosa26 does not disrupt neighboring genes. Expression from Rosa26 is consistent across generations without position effect variegation. These properties make Rosa26 superior to random integration approaches.
Rosa26 Targeting Applications
Common applications include: constitutive transgene expression using CAG or Rosa26 promoter; conditional expression using lox-STOP-lox cassettes; tissue-specific Cre or CreERT2 driver lines; reporter alleles for lineage tracing; inducible systems (Tet-On/Tet-Off); and humanization of cytokines or receptors requiring ubiquitous expression.
Frequently Asked Questions
What promoters are used for Rosa26 transgene expression?
The CAG promoter (hybrid CMV enhancer/chicken beta-actin promoter) is most commonly used for strong, ubiquitous expression from Rosa26. The endogenous Rosa26 promoter provides moderate, ubiquitous expression. Tissue-specific promoters can also be used at Rosa26 for defined expression patterns without position effects.
How does Rosa26 targeting compare to pronuclear injection?
Rosa26 targeting provides single-copy integration at a defined location with predictable expression levels, while pronuclear injection produces random multi-copy integration with variable expression and potential insertional mutagenesis. Rosa26 is preferred when consistent expression, defined copy number, and germline stability are required.
Are there other safe harbor loci besides Rosa26?
Yes. The H11 (Hipp11) locus on chromosome 11 and Hprt locus on the X chromosome are alternative safe harbors. Col1a1 is also used for some applications. However, Rosa26 remains the most widely used due to extensive validation, ubiquitous expression, autosomal location (avoiding X-linked sex effects), and broad community adoption.
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