Fig. 1
Generation of Mid1 conditional knockout in NCCs. A Schematic diagram of the construction of the Mid1-floxed allele for generating the conditional knockout of Mid1 in the NCCs. Exon 5 of Mid1 was flanked by loxP sites. The FRT flanked Neo cassette was deleted by crossing chimeras with FLP females generated the Mid1-floxed F1 heterozygotes (Mid1flox/+), which were subsequently crossed with Wnt1-Cre transgenic mice to generate Mid1-cKO male mice (Mid1flox/y; Wnt1-Cre). The position and direction of primers used for genotyping were indicated with black arrows. B The body weight (left) and body length (right) have been monitored weekly in Mid1+/y, Mid1flox/y, and Mid1flox/y; Wnt1-Cre mice. C PCR genotyping. Fragments of IL3 (544 bp) and Sry (402 bp, male-specific) were amplified for determination the gender of the mice. A 350 bp Cre replicon was amplified from the expression cassette of the Cre recombinase. Allele-specific PCR amplified 499 bp wild-type and 557 bp loxP fragments at the 5’ end loxP site to identify the F1 heterozygous. Allele-specific PCR amplified 344 bp wide-type and 466 bp loxP amplicons at the 3’ end loxP site to determine the heterozygosity at this locus. D PCR testing for identifying the deletion of Mid1 exon 5. A 1721 bp wild-type and a 511 bp knockout fragments were amplified using genomic DNA of Mid1-cKO mice. E RT-PCR using the cDNA of a Mid1-cKO male mice, showing a 173 bp amplicon of the transcript from Mid1 mutant allele in target tissue and a 310 bp amplicon of the transcript from intact Mid1 allele. F Western blot detected a 75-kDa MID1 protein in wild-type male mice and a reduced expression in Mid1-cKO male mice. Protein levels were normalized with respect to the level of β-actin expression. G Immunofluorescence of transverse sections of neural tubes (nb) of E10.5 embryo showed the ablation of Mid1 expression in the neural crest cells of Mid1-cKO mice. The migrating NCCs were identified with anti-Sox9 (green) and the expression of Mid1 was identified with anti-MID1 (red)