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.
“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.”– Hamid M. Said, PhD, PharmD University of California, Irvine
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.
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.
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.
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.
Pictured above 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.
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 Adv9(23): eadf3525.
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 Immunol210(11): 1790-1803.
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 Commun14(1): 972.
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.
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.
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.
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