Supplementary Information
Supplementary Table 1
Wt/Wt
Wt/Wt
Wt/CT
Wt/CT
CT/CT
CT/CT
Wt/p53–
p53–/p53–
Wt/p53–
p53–/p53–
Wt/p53–
p53–/p53–
normal (1)
normal (2)
normal (10)
normal (3)
abnormal (3)
abnormal (3)
Genetic ablation of p53 does not rescue embryonic lethality in Dido3CT-RFP mice.
Intercrosses between heterozygous female Dido3CT-RFP mice lacking one copy of p53
(Wt/CT; Wt/p53–) and male p53 knockout mice heterozygous for Dido3CT-RFP (Wt/CT;
p53–/p53–) yielded no viable pups homozygous for the Dido3 mutation (CT/CT). Embryos
from three intercrosses were analyzed for morphological abnormalities at E8.5. Numbers in
parentheses indicate the number of embryos analyzed.
1
Supplementary Figure 1
To generate a Dido3-specific loss-of-function mouse mutant, we constructed a targeting vector
that replaced exon 16 of the Dido locus with a red fluorescence protein (RFP) cassette in-frame
(top), which specifically disrupts expression of the Dido3 isoform after homologous
recombination in murine ES cells (Dido3CT-RFP).
The targeting construct was made by
bacterial artificial chromosome (BAC) recombineering. Briefly, we used a 72-base primer with
51 bases identical to the sequence of Dido3 exon 16, into which we introduced the RFP cassette
in-frame and 21 bases identical to the RFP cassette. The second primer was also 72 bases long,
with 51 bases identical to the Dido3 3’-UTR and 21 identical to the neomycin/kanamycin
cassette. We amplified the product by PCR with proofreading Taq and transfected the product
into the DY380 E. coli strain containing the pBeloBac with the Dido gene and a thermosensitive
phage. After thermoinduction and antibiotic selection, we obtained the recombined BAC clone.
We repeated the method with different primers to obtain a targeting vector by gap repair. R1 ES
cells were electroporated with the linearized vector; G418- and gancyclovir-resistant ES cell
clones were isolated and analyzed. Genomic Southern blotting (bottom) confirmed correct and
single insertion of the vector in the ES cell clone.
2
Supplementary Figure 2
Sections from Wt/Wt and CT/CT embryos at E7.5 were assayed for Dido3 expression (Dido3positive cells, red dots). Nuclei were DAPI-counterstained (blue). Dido3 is expressed in Wt/Wt
embryos; CT/CT embryos do not stain for Dido3. Red dots outside the embryo are indicative
of Dido3 expression in heterozygous maternal tissue. Arrowheads indicate presence (Wt/Wt) or
absence (CT/CT) of Dido3 expression in the embryonic ectoderm.
3