Custom cDNA Knockins
Precisely express your sequence of interest in a mouse model
The cDNA knockin approach offered by ingenious targeting laboratory provides researchers with a powerful tool to precisely express specific sequences of interest in mouse models.
By leveraging the regulatory elements of the endogenous gene, such as the promoter, the 5' UTR, and the chromosomal environment, this strategy enables the stable expression of a new sequence, including human cDNA, tissue-specific expression of Cre, a reporter gene, and more.
One of the key advantages of ingenious targeting laboratory's cDNA knockin method is its versatility. Researchers have the option to express the cDNA sequence in place of the target mouse gene or alongside it, depending on the experimental requirements.
Additionally, ingenious offers the possibility to design conditional and reversible cDNA knockin models by incorporating LoxP sites, providing even greater control over the expression of the inserted sequence.
What Researchers Say
"Our project manager did an outstanding job and has provided us with excellent customer service. Her availability to clarify issues has been nothing short of fantastic. I have recommended ingenious to others. Look forward for further collaboration on other projects."
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— Hamid M. Said, PhD, PharmD, University of California, Irvine
Custom cDNA Knockins Overview
Expressing a specific sequence in place of a target gene, or in addition to that gene, is a powerful and versatile strategy. A cDNA mouse knockin model allows expression of a new sequence using most regulatory features of an endogenous gene: the upstream promoter, the 5' UTR and the chromosomal environment.
This strategy allows expression of human sequence in place of mouse, tissue-specific expression of Cre or a reporter gene, and more.
We can also make your cDNA expression conditional by including LoxP sites in the design. Contact us to discuss available options.
From single point mutations to the most intricate designs ingenious has delivered custom knockin mice again and again for over 20 years. With a complete toolkit of different approaches and an experienced scientific staff, there is no knockin design that is out of reach.
Contact us today to discuss the custom knockin mouse model your lab will use for years to come.
cDNA Knockin to Replace Expression of Target Gene
This common mouse gene targeting approach accomplishes two objectives simultaneously: cDNA expression using a target gene's promoter, and knockout of that gene.
A cDNA knockin mouse model with a targeted insertion of Cre or a reporter gene will more faithfully match the expression pattern of the target gene, compared with transgenic strategies that only use a small promoter fragment.
If you need to express a sequence with many differences from the wild-type mouse sequence – such as a human gene sequence – then the cDNA knockin strategy is an excellent choice.
Applications:
- Create the most accurate reporter lines
- New Cre lines for your tissue of interest
- Express mutant and/or human sequence in place of wild-type mouse sequence
cDNA Knockin for Co-Expression with Target Gene
With precision gene-targeted knockins it's also possible to co-express a new gene along with a targeted gene of interest. Rather than replacing the target gene, both the cDNA knockin and the target gene will be expressed.
This is enabled by the IRES and 2A sequences which allow the production of two separate protein products from a single mRNA.
Using this strategy allows creation of reporter lines, Cre lines, and more, while retaining expression of the target gene.
Expression levels comparison:
| Method | Expression Levels |
|--------|-------------------|
| IRES | 2nd gene (after IRES) expressed at lower level than 1st |
| 2A | Expression level of both genes very similar |
Sequence changes comparison:
| Method | Sequence Changes |
|--------|------------------|
| IRES | IRES adds 500bp to mRNA, no change to protein sequence |
| 2A | 2A adds 63bp to mRNA, adds 20 amino acids to C-terminus of 1st protein and 1 proline to N-terminus of 2nd protein |
Conditional+Reversible Design
If you need a model that goes beyond standard available options, consider ingenious' conditional+reversible design. By giving you more control and allowing you to switch your gene's expression multiple times, this design can be used in a broad range of disciplines and applications.
This is an example of this strategy, where cDNA #1 is initially expressed in place of wild-type (WT). Activating Cre then leads to the expression of cDNA #2. When cDNA #1 and cDNA #2 are both deleted, the design reverts back to expressing the original WT sequence.
Client Publications Using cDNA Knockin
Kouvaros S, et al. 2023
Kouvaros S, Bizup B, Solis O, Kumar M, Ventriglia E, Curry FP, Michaelides M, Tzounopoulos T. 2023. A CRE/DRE dual recombinase transgenic mouse reveals synaptic zinc-mediated thalamocortical neuromodulation. Sci Adv 9 (23): eadf3525.
Harel M, et al. 2023
Harel M, Fauteux-Daniel S, Rodriguez E, Palmer G, Gabay C. 2023. IL-18 Binding Protein-Producing Cells Attenuate Anemia in Murine Macrophage Activation Syndrome. J Immunol 210 (11): 1790-1803.
Tebbe L, et al. 2023
Tebbe L, Mwoyosvi ML, Crane R, Makia MS, Kakakhel M, Cosgrove D, Al-Ubaidi MR, Naash MI. 2023. The usherin mutation c.2299delG leads to its mislocalization and disrupts interactions with whirlin and VLGR1. Nat Commun 14 (1): 972.
Frequently Asked Questions
What is cDNA and how is it used?
DNA is composed of both coding and non-coding sequences, whereas cDNA (or copy DNA) contains only coding sequences. cDNA is used as a tool in gene cloning and other research experiments.
What change is made to DNA in knock-in mice?
Gene knockin technology alters the genetic locus of interest via a one-for-one substitution of DNA sequence by the addition of sequence information that is not found on the genetic locus.
What happens to the DNA in Knockin mice?
Knockin mice may have a new genetic sequence added that is turned on in all cells, or only certain cells, or in response to mice receiving a specific drug. The knockin gene might cause a disease in the mice, or correct a problem caused by a different mutation, or mark certain cells with a fluorescent protein label.
Related Pages
- Knockin Mouse Models
- Point Mutation Mouse Models
- Humanized Mouse Models