I am using a company to make a knock-in mutant mouse. The company suggested the FLEx strategy (Cre on). How is it better than the traditional LSL strategy?
Hello Cheng-Ying,
the advantage of the FLEx design is that it allows you to keep the gene in which you knock-in the reporter functional until you conditionally activate the reporter expression. So it would be useful in case you work with an embryonic lethal gene inactivation and you need to use the reporter in homozygosity. In fact, the LSL construct can also be activated conditionally (depending on the Cre line you will use), but the KI per se will replace the wt gene from the START codon, needing the endogenous promoter for its expression, and will only be translationally silent until you delete the STOP codon with the recombinase, while the mRNA will be expressed accordingly to the endogenous promoter expression pattern.
The FLEx strategy was introduced in a 2003 Nature Biotechnology paper I attach below.
My recommendation in case you should choose the FLEx strategy, missing in the paper, would be to flank the Neo cassette with Frt sites (recombined by the Flp recombinase) because often the strong promoter placed in front of the Neo resistance gene sequesters transcription factors and de facto inactivates the expression of the endogenous gene, so it is always better to remove it.
Still, most times KI strategies produce hypomorphic alleles, so don't be surprised to see reduced expression of the wt gene as well as the reporter gene.
Last point, also not present in the paper, many times the fusion of the reporter gene to the beginning of the wt protein inactivates the function of the reporter (fluorescence or enzymatic activity), therefore it is safer to use an IRES (internal ribosome entry site) after the splice acceptor (SA) site, so to have independent translation of the reporter from a hybrid mRNA that fuses the initial exons of the wt gene to the reporter.
In general if you plan to use the KI mouse in heterozygosity (or the inactivation of gene you will knock the reporter in is not lethal for the mouse) the LSL approach is definitely simpler to design!
I hope this helps to make your choice. Good luck and best regards.
Thanks for the answer. You are right. I think suspected hypomorphism due to leaky mutant expression may be the reason why the company chose this strategy. It is not the typical FLEx but the DIO (double inverted open-reading-frame). It is a disease mutation KI. No reporter.