Transgenic Mice

When considering the creation of transgenic mice, one can choose a random integration approach via pronuclear injection, or a more precise and predictable gene targeted approach using a “safe harbor locus” such as Rosa26. While the random integration approach may be a faster way to generate an over-expression mouse model, numerous founder mice need to be produced and assayed to identify proper transgene expression. Due to the random nature of the integration, multiple copies of the transgene can integrate into the genome resulting in a higher likelihood of undesired gene disruptions. The targeted approach is a more precise way of inserting a transgene into the mouse genome and the mice will contain single and stable integration of the transgene at a transcriptionally active gene locus.

Targeted Transgenic Mice Using the Rosa26 Locus

As an alternative to random integration of the transgene of interest, which can be unpredictable and may perturb endogenous gene activity [1], the transgene can be inserted into a specific genetic locus for expression. There are several “safe harbor” loci that have been identified for “targeted transgenic” insertion of a transgene, including Rosa26, Hprt, and H11, to name a few [1]. Here, we discuss creating targeted transgenic mice via knockin at the Rosa26 locus. This insertion is not expected to cause unwanted phenotype resulting from modification of the Rosa26 locus. Transgenic mice utilizing the Rosa26 locus have been extensively published and validated.

At ingenious, we utilize our Rapid Rosa26™ Targeting technology for production of targeted transgenic models in shorter production timelines and at reduced costs, 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. Below are examples of some of the models we can create utilizing Rapid Rosa26™ targeting:

Ubiquitous Expression from Rosa26 Promoter

A transgene can simply be inserted into the Rosa26 locus for ubiquitous expression or 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.

Ubiquitous Expression from Rosa26 Promoter from


Tissue Specific or Temporally Controlled Expression Using a Floxed Stop Cassette

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. The use of this design involves mating the transgenic mice to Cre or CreERT2 mice, and it is important to factor that into the work and time needed to produce a study-ready mouse colony. The image below depicts the above described Rosa26 construct. Note that reporter genes or tags can be included with the cDNA of choice.

Tissue Specific or Temporally Controlled Expression Using a Floxed Stop Cassette from


Precision Control with ingenious’ Inducible Rosa26-Express™ Targeting System

Inducible Rosa26 Express Transgenic Mice from

One way to obtain transgenic mice with tissue specific and doxycycline inducible/reversible control in the Rosa26 locus, is to utilize ingenious’ Inducible Rosa26-Express™ targeting system. By designing a targeting vector that contains a floxed stop cassette and all of the necessary Tet-On components, we enable inducibility and tissue-specificity without the need for transactivator or silencer lines. Simply mate the targeted transgenic mice with a tissue-specific Cre recombinase line (of which hundreds are available in repositories for you to select from), and introduce doxycycline for inducible/reversible expression of your transgene in your tissue of interest. Click to read more about our Inducible Rosa26-Express™ Targeting System.


Inducible Regulation of Gene Expression Using ingenious’ F.A.S.T.™ Technology

Another way to obtain inducible and reversible control of your transgene at the Rosa26 locus is by use of ingenious’ F.A.S.T.™ technology. The F.A.S.T.™ system provides a variety of expression options including global gene inactivation, tissue/time-specific gene expression, ectopic gene expression, inducible and reversible gene overexpression, and inducible and reversible gene knockdown, depending on which transactivator or transilencer is used in combination with the F.A.S.T.™ system. Our F.A.S.T.™ cassette can also be modified to include a reporter, to leave out the stop component, or to have other modifications that fit with your specific research goals. Click to read more about our inducible & reversible F.A.S.T.™ system.

Inducible Regulation of Gene Expression Using ingenious’ F.A.S.T. Technology from


In summary, utilizing a safe harbor locus to produce a targeted transgenic model allows for:

  • Predictable transgene integration.

  • Single copy number insertion.

  • Presence of Rosa26 cis-regulatory elements.

  • Less mice need to be screened to find a suitable germline founder – More 3R’s friendly.

  • Using ingenious’ technologies to speed up the model production process, saving you time and money when compared to standard knockin models.

  • Sophisticated design options, making your model more versatile.


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Transgenic Mice Using Random Integration

Traditional transgenic mice are generated by introducing a simple DNA fragment into fertilized mouse oocytes via pronuclear injection. The DNA fragment is typically a mouse or human cDNA / gene, driven by a specific promoter. The purpose of transgenic mice is to over-express a mouse gene of interest, or express a foreign gene in the mouse, such as a human gene for humanized mouse models.

Process Details and Considerations

After DNA fragments are generated and injected into mouse oocytes, founder mice are born which then need to be genotyped for integration of the transgene. This process, from concept to mice can take about 6 months [2]. Although the timeline for production of transgenic mice is relatively short, care must be taken to evaluate location of the insertion to ensure that no other gene has been disrupted by the insertion. Furthermore, multiple copies can randomly integrate into the genome and it may be necessary to screen a large number of mice until the appropriate founder has been identified. This founder will contain the desired number of copies and expression levels, without any other gene having been disrupted by the integration(s). In addition, the use of smaller transgenes can lead to position-mediated silencing and ectopic expression following transgene integration.

Because of these limitations, the use of BAC transgenes may be favored [3]. BACs can hold up to 200kb of sequence, which allows for large sequences such as entire genes or large promoters to be introduced into the mouse. Inserting an entire gene including introns, exons and regulatory sequences is beneficial when the coding sequence alone may not be sufficient for proper expression or research study. The use of BAC transgenic constructs can result in more faithful gene regulation and processing as well as the potential to perform functional and regulatory studies on multiple transcripts.

Things to keep in mind when deciding if a pronuclear injection transgenic model is right for you:

  • Random and unpredictable transgene integration and expression.

  • Variable copy numbers of the transgene.

  • Small transgenes may lack the cis-regulatory elements needed for proper expression [1].

  • BAC constructs are harder to work with and require more molecular biology expertise.

  • Need to screen founder mice (as opposed to ES cells). May not be 3Rs-friendly [4].

After You Receive Your Mice

ingenious offers an array of targeted transgenic solutions to meet your research needs. In particular, our exclusively licensed F.A.S.T.™ technology, Rapid-Rosa26™ and Inducible Rosa26-Express™ systems require additional mating to unlock their full gene regulation potential. ingenious’ post project support services makes using our models easy and provides an accurate path to the generation of cohorts, as well as cryopreservation services to protect your mouse lines.


1) Sadelain, Michel, Eirini P. Papapetrou, and Frederic D. Bushman.Safe Harbours for the Integration of New DNA in the Human Genome.Nature Reviews Cancer 2012 12(1): 51-58.

2) Timeline for Transgenic Mice Analysis, University of Michigan

3) BAC recombineering and transgenesis, Vanderbilt University

4) What are the 3Rs? NC3RS