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Transgenic Mice

Transgenic mice have proven to be an invaluable research tool for studying human disease. Initially, lines were generated by pronuclear injection which results in random integration of the transgene into the genome. Initially, lines were generated by pronuclear injection, which results in random integration of the transgene into the genome. Two advances improved the reliability of these models: cultured embryonic stem cells and pioneering work demonstrating the advantages of “safe harbor” loci such as Rosa26 for stable and reliable integration. Compared with random integration, a targeted insertion into a safe harbor locus is more likely to have a single insertion with predictable expression levels. The risk of the transgene interrupting endogenous mouse genes is eliminated and transgene expression won’t be disrupted by surrounding genetic elements. Scientists at ingenious have extensive experience targeting the Rosa26 locus – contact us today to discuss the perfect targeted transgenic line for your human disease research.

Utilizing ingenious’ technologies allows for:

Predictable transgene integration.

Single copy number insertion.

Efficient identification of germline founders – more 3R’s friendly.

Rapid model production, saving you time and money when compared to standard knockin models.

Sophisticated design options, making your model more versatile

Targeted Transgenic Mice Using the Rosa26 Locus

At ingenious, we utilize our Rapid Rosa26™ Targeting technology for production of targeted transgenic mice with shorter production timelines and 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. No design is too difficult for ingenious – contact us today to discuss a new transgenic line with a large insert or complex genetic elements.

Below are examples of some of the models we can create utilizing Rapid Rosa26™ targeting:

Ubiquitous Expression

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.

transgenic mice

 

Tissue Specific or Temporal Control

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.

Inducible Rosa26-Express™ Targeting

One way to obtain transgenic mice with tissue specific and inducible control 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.

 

New options for overexpression mouse models are making  Rosa26 lines more versatile than ever. Create a flexible model with temporal and spatial control of your gene of interest using ingenious’ Inducible Rosa26-ExpressTM targeting system. By combining tissue-specific Cre abilities with drug inducibility all in one model, our technology gives you the precise control over gene expression that you need.

Inducible Rosa26-Express™ Targeting Benefits

  • Inducible and tissue-specific transgene control with combined regulation by Cre and TetO
  • Reduced time and cost: Our Inducible Rosa26-Express™ is combined with our Rapid-Rosa26™ Targeting technology to reduce the time and cost required to generate your transgenic mouse model.
  • Safe-harbor Rosa26 locus: predictable activity with no risk of disrupting other genes

Flexible Overexpression with Rosa26-Express™ from ingenious

ingenious has taken the complicated process of generating tissue-specific inducible transgene expression systems and simplified it. You control expression of your transgene by crossing your new Rosa26-express line with any Cre-expressing line. All the elements necessary for inducible expression are present – crossing with a Tet-expression line is not necessary.

 

 

The design works by incorporating a reverse tetracycline-controlled transactivator (rtTA) component, a TRE3G (tetO) promoter, and a floxed Neomycin-Stop cassette into the “safe harbor” Rosa26 locus. The Neomycin-Stop cassette, inserted downstream of the CAG promoter, prevents rtTA expression, which is one of the key components required for transgene expression from the TRE3G promoter.

 

Mating the Inducible Rosa26-Express™ mouse line to a ubiquitous, or tissue-specific Cre recombinase mouse line allows for removal of the floxed Neomycin-Stop cassette, for expression of rtTA from the pCAG promoter.

 

 

Doxycycline introduced to the mouse via food, water, or injection, binds with high affinity to the rtTA, allowing it in turn to bind to the TRE3G promoter and activate expression of the transgene. Removal of doxycycline renders the rtTA unable to bind to the TRE3G promoter and expression stops.

 

Resources for Further Reading

1) Rapid-Rosa26™ Targeting technology from ingenious

2) Tet Systems: principles and components

3) Tet Systems: home page

4) Schönig K et al. (2010) The power of reversibility: regulating gene activities via tetracycline-controlled transcription. Methods Enzymol. 477: 429-453.

5) Stieger K et al. (2009) In vivo gene regulation using tetracycline-regulatable systems. Adv Drug Deliv Rev 61(7-8): 527-541.

F.A.S.T.™ Targeting

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 trans-silencer 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.


Transgenic Mouse Models Using Random Integration

Traditional transgenic mouse models are generated by introducing DNA into fertilized mouse oocytes via pronuclear injection. The DNA fragment is typically a mouse or human cDNA, driven by a specific promoter, with the goal of over-expressing a gene of interest. This method has generated many invaluable models but does entail a few drawbacks that must be accounted for.

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Process Details and Considerations

Pronuclear injection of DNA can lead to low frequency integration of the sequence into the mouse genome at random sites. After potential founder mice are born they must be screened for germline transmission of the desired sequence. Ideally multiple alleles will be evaluated as expression of the transgene can be affected by its genomic position, and other genes can be disrupted if the transgene inserts within their sequence. Furthermore, multiple copies can randomly integrate into the genome- it may be necessary to screen a large number of mice until the appropriate founder has been identified.

In some cases the desired DNA sequence can be injected as a bacterial artificial chromosome (BAC) to alleviate some concerns with random insertion transgenesis. 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 of your human disease 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].

Consider targeted transgenics

Targeted transgenic approaches reduce or eliminate the downsides of random integration by pronuclear injection. The safe-harbor Rosa26 locus can be targeted by ingenious’ Rapid-Rosa26™ strategy to efficiently introduce your overexpression construct into the genome in a location where it will not interfere with other genes or be affected by the surrounding genetic environment. To learn more about transgenic mice, speak with one of our scientific consultants today. 

References

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



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