Fig. 6. Targeted knock-in of Emx2 cDNA into the Otx2 locus. (A) Diagrammatic representation of knock-in strategy. Thick lines represent Otx2 genomic sequences; thin lines, pBluescript sequences; black boxes, Otx2 coding exons; black triangles, loxP sequences. White boxes denoted as Em, neo^r and DT-A indicate Emx2 cDNA lacking 3'UTR, neomycin-resistant gene lacking polyadenylation signal driven by Pgk1 promoter and diphtheria toxin-A fragment gene with MCI promoter, respectively. S is the probe for Southern blotting used to identify homologous recombinant ES cells shown in B; p1 and p2 are PCR primers used in the detection of the homologous recombinants in ES cells, and p3 and p4 are PCR primers used in the detection of the deletion of the neo^r cassette in F₁ heterozygotes. H, HindIII; N, NotI; Nl, NlaIII; S, SalI; Sm, SmaI; Sp, SphI. (B) The top panel displays an example of Southern blotting used to identify homologous recombinants in ES cells; the bottom panel displays an example of the PCR genotyping for the deletion of neo^r cassette in F₁ offspring obtained by mating between male chimera and Cre females. (C,D) RNA expressions from both endogenous and knocked-in Emx2 at 10.5 dpc in a wild-type embryo (C) and a knock-in mutant (D). Emx2 is expressed in the caudal diencephalon and mesencephalon where endogenous Emx2 is absent. (E,F) EMX2 protein expressions at 10.5 dpc in a wild-type (E) and a knock-in embryo (F). Consistent with the ectopic mRNA expression, EMX2 protein is detected in the caudal diencephalon and mesencephalon. The endogenous Otx2 expression and thus the knocked-in Emx2 expression is somewhat weaker in the midbrain than in the forebrain at this stage. (G,H) Enlarged views of E,F (respectively) at the telodiencephalicus level indicated by arrows. Arrowheads indicate the isthmus. Scale bars: 125 µm.