Generation of <i>Dmxl2</i> KO mice.

(A) Genetic construct. Dmxl2 is located on chromosome 15 in mice, and encodes 9 isoforms, 4 of which are reported to be protein-coding in Ensembl (ENSMUSG00000041268). The longest form is represented here; it has 44 exons containing 13 WD40 domains according to SMART (exons encoding WD40-domains are shown in gray). Heterozygous mice (Dmxl2wt/tm1a) were obtained from the International Mouse Phenotype Consortium http://www.mousephenotype.org. The mutation corresponded to the insertion of the targeting vector between Dmxl2 exons 6 and 10 (red line). The targeting vector contained a splice acceptor site and an IRES in Dmxl2 intron 6 upstream from the LacZ reporter gene, and a polyadenylation signal downstream from LacZ. Consequently, Dmxl2 transcripts truncated after exon 6 should be produced and LacZ should be expressed under the control of the endogenous Dmxl2 promoter. FRT: flippase recognition target; En2 SA: splice acceptor of mouse Engrailed2 exon 2; IRES: internal ribosome entry site; LacZ: gene encoding β-galactosidase; pA: polyadenylation signal; loxP: sequence targeted by Cre recombinase; hBactP: human β-actin gene promotor; Neo: neomycin resistance gene. (B) PCR genotyping on mouse DNA yielded the following amplicon sizes: 211 bp for the wild type; 211 and 189 bp for heterozygous mice; and 189 bp for KO mice. The two amplicons are indicated by different symbols as their sizes on the gel are very similar (¤ and * for 211 bp and 189 bp, respectively). (C) Potential residual expression of the Dmxl2 wild-type sequence in tissues with a knockout (KO) of the gene was assessed by PCR. The primers “a” and “b” used for this purpose are represented by blue arrows on the genetic construct diagram. No amplification was obtained with brain cDNA from KO mice. (D) Western blotting detected none of the four protein isoforms (341, 338, 249 and 146 kDa), confirming the efficacy of Dmxl2 invalidation in KO animals and antibody specificity. (E) Gross morphology of newborn knockout (KO) and heterozygous (HTZ) mice; note the empty stomach of KO animals.