Expressing a specific sequence in place of a target gene, or in addition to that gene, is a powerful and versatile strategy. cDNA knockin 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.
This common approach accomplishes two objectives simultaneously: cDNA expression using a target gene’s promoter, and knockout of that gene. A cDNA knockin 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.
|Expression levels||2nd gene (after IRES) expressed at lower level than 1st||Expression level of both genes very similar|
|Sequence changes||IRES adds 500bp to mRNA, no change to protein sequence||2A adds 63bp to mRNA, adds 20 amino acids to C-terminus of 1st protein and 1 proline to N-terminus of 2nd protein|
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.
1) Vagner S, Galy B, Pyronnet S. 2001. Irresistible IRES. Attracting the translation machinery to internal ribosome entry sites. EMBO Rep 2(10): 893-8.
2) Kim JH, Lee SR, Li LH, Park HJ, Park JH, Lee KY, Kim MK, Shin BA, Choi SY. 2011. High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. PLoS One 6(4): e18556.