Figure 6. Ac45-Flox^Neo mice with neomycin is embryonic lethal.

(A) Strategy of generating Ac45-Flox^Neo mice. The Ac45-Flox^Neo mice was generated by inserting two loxP sites in the intron upstream of exon 3 and another in the intron downstream of exon 4, with a neomycin-resistance (neo) expression cassette inserted as a selection marker. (B) Southern blot of six ES clones (1–6) and a negative control. 5′-SphI digest screening reveals WT and KO band size of 14.8 and 7.1kB respectively. 3′-SphI digest screening reveals WT and KO band size of 14.8 and 6.9kB respectively. (C) Tail-tip genotyping of the embryos demonstrates that clones 1 to 5 were Ac45-Flox^Neo embryo (Ac45_FRT_tR1 and Neo3F primers produce a product size of 429bp for floxed allele and no product for wildtype) while clone 6 is the WT embryo (Ac45_GF2 and Ac45_FRT_tR1 primers produce a product size of 237bp for wildtype and no product for floxed). (D and E) Immunohistochemistry demonstrated Ac45 expression is ubiquitous and high in brain and skin in the WT embryo whereas Ac45 expression was not detected in the Ac45-Flox^Neo embryo. In addition, the Ac45-Flox^Neo embryo had ventricular enlargement and neuronal loss, demonstrating impairment of the brain development (arrows).