Rapid-Rosa26™ Targeting technology is ingenious’ solution to the call for custom targeted transgenic models with shorter production timelines and reduced cost, without compromising performance and quality. We utilize well-validated gene targeting strategies combined with our ready-to-go enhancing vectors to deliver your expression cassette or cDNA to the Rosa26 locus in record time.
Do you have another safe harbor locus in mind to use for your mouse model other than Rosa26? Contact us to discuss your goals and the design you are interested in developing for your research.
No design is too difficult for ingenious – contact us today to discuss a new transgenic line with a large insert or complex genetic elements.
The Rosa26 locus in the mouse genome defines an animal model in which a transgene is inserted in a permissive locus, also known as a “safe harbor” which allows researchers to express genes of interest. Different gene targeting technologies (embryonic stem cells, CRISPR) are used to make specific DNA insertions at the Rosa26 locus. The transgene can be constitutively or inducibly overexpressed.
Targeting transgenes to a chosen location in the genome has a number of advantages. A single copy of the DNA construct can be inserted by targeting regions of chromatin that allow the desired development and tissue-specific expression of the transgene. The Rosa26 locus is such a genomic site that is ideal to integrate a transgene of interest for stable expression.
Below are examples of some of the models we can create utilizing Rapid-Rosa26™ Targeting:
A transgene can simply be inserted into the Rosa26 locus for ubiquitous over-expression by means of the Rosa26 gene promoter or CAG promoter. The CAG promoter is a strong, ubiquitously expressing promoter which can be utilized when strong over-expression is desired.
The use of a loxP flanked stop cassette will enable you to activate gene expression in your tissue of choice through mating with a tissue-specific Cre mouse line. Or, CreERT2 mice can be utilized to provide inducible transgene expression upon treatment with Tamoxifen. This approach creates a versatile line which can overexpress your construct in any tissue for which you have a Cre line.
One way to obtain transgenic mice with tissue specific and inducible control is to utilize ingenious’ Inducible Rosa26-Express™ targeting system. By combining Cre abilities with drug inducibility all in one model, our technology gives you the precise control over gene expression that you need. Click here for more information on our Inducible Rosa26-Express™ system.
Located on the mouse chromosome 6, the Rosa26 locus spans around 9KB and consists of three exons. It is moderately expressed with levels varying between different tissues in pre and post natal life. Currently, Rosa26 is a safe harbor locus broadly used for both constitutive and conditional gene expression in mice. It is proven safe in mice because the transgene is adequately expressed without perturbing endogenous gene structure or function.
Generally, there are three different types of Rosa26 locus knockins. In the original design, the transgenic cDNA is expressed constitutively from the Rosa26 promoter. Specifically, a splice acceptor (SA) sequence followed by cDNA of the transgene of interest is inserted into the respective restriction site within 1st intron of the Rosa26 gene. A stop cassette, composed of a neomycin resistance gene and polyadenylation (pA) sites, are inserted upstream of the transgene. A variation of the 1st strategy for conditional transgene expression is realized by using a stop cassette flanked by two lox P sites. In this case, the presence of a stop cassette can prevent the transgene from expressing unless the Cre is expressed to remove the stop cassette via homologous recombination.
If the strength of the Rosa26 promoter is not able to achieve the desired levels of transgene expression, an exogenous or enhanced promoter could be used. An example of this type of promoter is CAG and is introduced to drive high transgene expression from the Rosa26 locus.
Rosa26 locus targeting approach allows for a very robust, high frequency of correct insertion and stable expression that is widely used by the scientific community. ingenious has generated a fast, ready to use targeting vector with a variety of genetic designs that could be utilized for different applications. If tissue-specific control is essential, ingenious has designed a Rosa26 mouse line which contains a floxed stop cassette and all of the necessary Tet-On control components. This Rosa26 mouse line enables tissue-specificity and inducibility of your cDNA without the need for transactivator lines.