FIG. 4.

Homozygous inactivation of Dppa2 in ES cells lacking Dppa4. (A) Targeting strategy to inactivate both Dppa2 alleles in ES cells of homozygous Dppa4-deficient clone 248.44. Filled boxes, exons; red triangles, loxP sites; black triangles, FRT sites; TK, thymidine kinase cassette for selection against random integration events. After transient Cre expression, the loxP sites on one Dppa2 allele have recombined with the residual loxP site in the Dppa4 locus, deleting all sequences in between. (B) Targeting history of the two Dppa2/Dppa4 doubly deficient ES clones 30.15 and 30.18. All manipulations started with clone 248.44 already lacking Dppa4 (Fig. 1). Flox-Neo, allele on which exon 2 has been flanked by loxP sites (floxed) and an FRT-flanked Neo selection cassette inserted into intronic sequences upstream of exon 2; Flox, allele on which exon 2 has been flanked by loxP sites; ΔEx2, allele on which exon 2 has been deleted; ΔEx1+2, allele on which loxP sites flanking the Neo cassette have recombined with the residual loxP site in the Dppa4 locus, deleting exons 3 to 7 of Dppa4 and exons 1 and 2 of Dppa2. (C) RT-PCR to detect Dppa2 (top) and Dppa4 (bottom) transcripts in targeted ES clones. Note the absence of functional full-length Dppa2 message (upper band) in homozygous Dppa2-deficient (Δ/Δ) clones 30.12, 30.15, 30.28, and 30.31. In these clones, Dppa4 message is also missing due to insertion of the tdRFP cassette into one Dppa4 allele and the Cre-mediated recombination event between Dppa2 and Dppa4 loci on the other allele. The Dppa4 genotypes and history of clones 84.2 and 248.44, in which the Dppa2 locus is unmanipulated are shown in Fig. 1B. Clones 34.5 and 34.8 were generated by transient expression of Cre recombinase in clone 34 to obtain heterozygous Dppa2^+/− control cells. (D) Immunoblot of protein extracts from the indicated ES cell clones demonstrating complete absence of Dppa2 protein in clones 30.15 and 30.18, which were derived from Dppa4-deficient clone 248.44. The polyclonal rabbit-anti-mouse Dppa2 serum used was directed against an epitope encoded by exon 7. The polyclonal rabbit anti-mouse Dppa4 serum used (lower panel) was the same as described for Fig. 1H. The filter was first hybridized with the anti-Dppa2 antiserum (top) and then stripped and rehybridized with an anti-alpha-ERK antibody (middle) as a loading control. Equal amounts of protein extract were run in parallel on a separate gel for the Dppa4 blot (bottom). The ES cell clones shown have the following Dppa2-Dppa4 genotypes: E14.1, wild type (WT)/WT-WT/WT; clone (Cl) 30.15, knockout (KO)/KO-KO/KO^RFP; Cl 30.18, KO/KO-KO/KO^RFP; Cl 180.5, WT/WT-Flox/WT; Cl 248.44, WT/WT-KO/KO^RFP. (E) Phase contrast microscopy of Dppa2/Dppa4 doubly deficient ES colonies documenting typical ES cell morphology (top) and homogenous expression of the pluripotency marker TNAP. Scale bars, 100 μm (top) and 200 μm (bottom). (F) Immunoblot showing expression of marker proteins (Sox2, Oct4, and Nanog) characteristic for pluripotent cells. (G) No significant difference in proliferation rates between Dppa2/Dppa4 doubly deficient ES cells (clones 30.15 and 30.18) and wild-type ancestor cells (clone 180.5, exon 2 on one Dppa4 allele floxed). ES cells were plated at 7.5 × 10³/ml in duplicate wells and counted at 24-h intervals for 96 h. (H) No significant difference in cell cycle kinetics between Dppa2/Dppa4 doubly deficient ES cells (clones 30.15 and 30.18) and wild-type control cells (clone 180.5). Cells were stained with PI and analyzed by flow cytometry. The percentage of cells in G₁, S, and G₂ phases was determined using the FlowJo cell cycle analysis software Dean/Jett/Fox. α, anti.