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. 2009 Mar 30;29(11):3186–3203. doi: 10.1128/MCB.01970-08

FIG. 8.

FIG. 8.

Dppa4-driven tdRFP expression in pluripotent cells. (A) Loss of tdRFP expression in ES cells upon in vitro differentiation. ES cells/embryoid bodies of clone 565 were harvested on the indicated days of differentiation, dissociated, and analyzed by flow cytometry for tdRFP and GFP expression. ES cells of this clone (derived from clone 167ΔNeo by targeting of GFP into the Brachyury locus) carry tdRFP in one Dppa4 allele and a GFP expression cassette in one allele of the Brachyury gene locus (19). GFP-Brachyury expression serves as a marker for cells that have adopted a mesodermal fate and thus for monitoring correct kinetics of in vitro differentiation. (B) tdRFP expression in preimplantation embryos. The embryos were obtained from timed matings between heterozygous females (WT/KORFP) and wild-type males (WT/WT). At early stages of development, also embryos with two wild-type alleles exhibit red fluorescence due to expression of maternally deposited tdRFP. The two-cell embryos (white arrowheads), which were obtained from a mating between wild-type C57BL/6 animals, were therefore included as negative control. Scale bar, 150 μm. (C) Strong tdRFP expression in primordial germ cells (PGCs) after colonization of both female and male genital ridges (E12.5). Note the normal number of tdRFP-labeled PGCs in homozygous Dppa4-deficient embryos. (D) PGCs expressing tdRFP can be easily identified and isolated by fluorescence-activated cell sorting. Dot plots show a flow cytometric analysis of cells obtained from dissociated genital ridges of E12.5 embryos. The gender of the animals was determined by PCR. Again, note the normal number of tdRFP-labeled PGCs in homozygous Dppa4-deficient embryos after staining with an anti-kit (anti-CD117) antibody. (E) Expression of tdRFP in testis is localized to developing sperm. Testes and epididymides are from newborn pups. (F) Reactivation of Dppa4-driven tdRFP expression in iPS cells generated from tdRFP/Dppa4 knock-in MEFs. MEFs heterozygous or homozygous for the tdRFP/Dppa4 mutation were transfected with retroviruses encoding the pluripotency-inducing factors OCT4, SOX2, and KLF4 (34). The middle and right panels show representative primary colonies 3 to 4 weeks after retroviral transduction of MEFs. Knock-in MEFs used for reprogramming experiments do not exhibit any red fluorescence (left panels). KO, knockout.