Up until recently, methods for generating floxed mice either conventionally or by CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9 (CRISPR-associated protein 9) editing have been technically challenging, expensive and error-prone, or time-consuming. To circumvent these issues, several labs have started successfully using a small artificial intron to conditionally knockout (KO) a gene of interest in mice. However, many other labs are having difficulty getting the technique to work. The key problem appears to be either a failure in achieving correct splicing after the introduction of the artificial intron into the gene or, just as crucial, insufficient functional KO of the gene's protein after Cre-induced removal of the intron's branchpoint. Presented here is a guide on how to choose an appropriate exon and where to place the recombinase-regulated artificial intron (rAI) in that exon to prevent disrupting normal gene splicing while maximizing mRNA degradation after recombinase treatment. The reasoning behind each step in the guide is also discussed. Following these recommendations should increase the success rate of this easy, new, and alternative technique for producing tissue-specific KO mice.
Keywords: artificial intron; mRNA degradation; splicing; splicing prediction program; tissue-specific knockout.