“Rosa26” (Symbol Gt(ROSA)26Sor) is a locus on mouse chromosome 6 that has been extensively used as a “safe harbor” insertion site for transgenes. The Rosa26 locus has an open chromatin environment permissive for transgene expression. The native Rosa26 locus expresses a long noncoding RNA (lncRNA) of unknown function, and disruption of the lncRNA expression does not cause any overt phenotype.

Transgenes are inserted in the Rosa26 locus by CRISPR targeting. This places the transgene in a well defined open chromatin environment that allows broad transgene expression with predictable characteristics. Transgenes may be inserted under the control of the native Rosa26 promoter for broad, moderate transgene expression. Alternatively, insertion of a transgene with the CAG promoter at the Rosa26 locus results in very strong, broad transgene expression. Our proprietary system allows the user to obtain both very strong and moderate expression levels from the same knockin model.

Transgenes are often inserted in the Rosa26 locus with a conditional element such as a loxP-flanked stop cassette between the promoter and the gene to be expressed. The stop cassette blocks expression until it is removed by Cre recombination. This conditional system allows the user to take advantage of the many existing, well-characterized Cre recombinase lines to drive tissue-specific transgene expression. The use of tamoxifen-inducible Cre can also allow temporal control of transgene expression. Our proprietary system allows inducible expression with two different expression levels.

Rosa26 targeted transgenesis provides a well-characterized, highly reproducible and predictable animal model. The initial model production timelines are similar between Rosa26 knockin and random transgenesis approaches. However, the defined, reproducible nature of the Rosa26 knockin system provides significant time and cost savings in the long-run compared to random transgenesis.

Random transgene integration is highly variable, with different copy numbers inserted at a random genomic location in each founder animal. The insertion site and copy number have significant effects on transgene expression, requiring the characterization of multiple founder lines to identify a line with desired characteristics. The random insertion may also disrupt a gene at the insertion site, introducing additional and unexpected phenotypic complexity. With the Rosa26 knockin system, a single copy of the transgene is inserted at the Rosa26 locus and only a single founder line is needed. The use of well-characterized transgene designs also provides high probability for successful expression.

Confirmed heterozygous knockin animals are typically delivered within 6-9 months.