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Researchers Recommend Verifying Gene Disruption in Knockout Mice Due to En2 Insertions

Posted on October 4, 2024

A recent study highlights the importance of confirming gene disruption in knockout mice that use the “knockout-first” allele design. This approach, developed by the International Knockout Mouse Consortium, incorporates the En2 splice acceptor sequence and the lacZ reporter gene to facilitate gene function studies. While intended to disrupt gene function by interfering with normal splicing, the En2 sequence can sometimes be included in the host gene’s mRNA. This inclusion may produce a mutant protein, resulting in partial gene activity rather than a complete knockout.

The researchers conducted a comprehensive computational analysis of over 14,000 mouse protein-coding genes with established knockout-first embryonic stem cell lines. They examined transcripts from 35 mutant lines and found that in 24 of these lines, the En2 sequence was present in the transcripts. This suggests that gene disruption might not be complete in these cases, potentially affecting the outcomes of experiments relying on these models.

Notably, the study did not find widespread effects on mouse phenotypes due to the predicted En2 insertion. However, the potential for partial gene activity underscores the need for researchers to verify the transcripts of their mutant mice. By confirming whether the En2 insertion is present, scientists can ensure that gene function is adequately disrupted, enhancing the reliability of their experimental results.

The study advises that mutant transcripts be checked for the En2 insertion and leaky transcription when using knockout-first alleles. This additional step can help researchers avoid unintended consequences and maintain the integrity of studies.

For more details, read the full study here: https://link.springer.com/article/10.1007/s00335-024-10071-2