Using homologous recombination in ES cells, the knock-in vector targeted exon 5 of the Ankfn1 locus and was integrated 21 bp into exon 5, replacing the remainder of the exon with a tdTomato-P2A-split Cre-Neo-WPREBGHpA cassette, which causes frameshifted nonsense mutations downstream, resulting in the loss-of-function allele. Neomycin was excised via FLP mice. (J:312000)