FIG. 1.
Homozygous inactivation of Dppa4 in ES cells and concomitant insertion of a red fluorescent reporter gene into one Dppa4 allele (Dppa4tdRFP) by successive gene targeting. (A) Targeting strategy used to generate homozygous Dppa4-deficient ES cells with one Dppa4tdRFP knock-in (KI) allele. Filled boxes, exons; red triangles, loxP sites; black triangles, FRT sites; TK, thymidine kinase cassette for selection against random integration events. (B) Targeting history of individual Dppa4-modified ES clones. Flox-Neo, allele on which exon 2 has been flanked by loxP sites (floxed) and a FRT-flanked Neo selection cassette inserted into intronic sequences upstream of exon 2; Flox, allele on which Dppa4 exon 2 has been flanked by loxP sites; tdRFP/ΔEx2-Neo, allele on which Dppa4 exon 2 has been replaced by a tdRFP expression cassette and a loxP-flanked Neo cassette; tdRFP/ΔEx2, allele on which Dppa4 exon 2 has been replaced by the tdRFP expression cassette; ΔEx2, allele on which Dppa4 exon 2 has been deleted. (C) Southern blot analysis of targeted ES clones described in panel B. Genomic DNA was doubly digested with BglII/XhoI and probed with a DNA fragment hybridizing downstream of exon 1 but outside the targeting construct. (D) RT-PCR analysis of total RNA from ES cells of the indicated clones with primers annealing to sequences encoded by the first and last Dppa4 exons (exon 1 and 7). The PCR product of 912 bp represents full-length Dppa4 message, while the product of 794 bp reveals absence of exon 2 and efficient splicing from exon 1 to exon 3 on the ΔEx2 allele. Note that insertion of the tdRFP expression cassette along with pA on the second Dppa4 allele blocks transcription beyond the insertion site and thus does not give rise to any PCR product with the specific primers used. (E) Sequence analysis of the splice junction in the 794-bp RT-PCR fragment shown in panel D. The nucleotide sequence confirms splicing between exon 1 and exon 3 on the Dppa4 knockout (KO) allele and the resultant shift in the reading frame, which leads to the appearance of an in-frame stop codon (TGA) after 33 amino acids (not shown). (F) Phase-contrast (top) and fluorescence (bottom) microscopy demonstrating that insertion of a tdRFP expression cassette in lieu of exon 2 on one Dppa4 allele results in specific labeling of targeted ES cells. The configuration of Dppa4 alleles is given in brackets. (G) Flow cytometric analysis of ES cells carrying one Dppa4tdRFP allele (clone 243.12) and a genetically unmanipulated control (E14.1). The histogram shows an overlay of the dot plots above. (H) Immunoblot of protein extracts from the indicated ES cell clones demonstrating absence of Dppa4 protein in clones carrying a KO and a KO/KI allele (clones 248.28, 248.44, 243.12, and 243.47). The polyclonal rabbit anti-mouse Dppa4 serum used is directed against an epitope encoded by exon 5. The signal in the MEFs lane is a nonspecific band of bigger size. WT, wild type.