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. 2017 Feb;53(2):179-190.
doi: 10.1007/s11626-016-0097-y. Epub 2016 Oct 3.

Expression Analysis of the Endogenous Zscan4 Locus and Its Coding Proteins in Mouse ES Cells and Preimplantation Embryos

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Free PMC article

Expression Analysis of the Endogenous Zscan4 Locus and Its Coding Proteins in Mouse ES Cells and Preimplantation Embryos

Kei-Ichiro Ishiguro et al. In Vitro Cell Dev Biol Anim. .
Free PMC article

Abstract

Mouse Zinc finger and SCAN domain containing 4 (Zscan4) is encoded in multiple copies of Zscan4 genes, which are expressed in late two-cell stage preimplantation embryos and in 1-5% of the embryonic stem (ES) cell population at a given time. Due to the highly identical nucleotide sequences of multiple copies of Zscan4 paralogs and pseudogenes in the mouse Zscan4 genomic cluster, previous analyses have been done using exogenous transgenes under the regulation of Zscan4c promoter. In this manuscript, we generated knock-in mouse ES cell lines and mouse lines, in which the expression of endogenous Zscan4c, one of the Zscan4 genes, can be specifically monitored with a green fluorescent protein variant, Emerald. Interestingly, we found that only ∼30% of Zscan4-immunopositive ES cells were Emerald positive, suggesting that even when the Zscan4 locus is active, not all Zscan4 genes are expressed synchronously. We also carried out mass spectrometry of protein complexes associated with endogenous Zscan4 proteins. Taken together, our genetic engineering at an endogenous Zscan4c gene provides the first clue for the expression and function of each gene copy of Zscan4 locus in a physiological context.

Keywords: ES cell; Knock-in; Preimplantation embryo; Two-cell stage; Zscan4.

Conflict of interest statement

M.S.H.K. is a cofounder of Elixirgen, LLC, which promotes clinical application of human Zscan4.

Figures

Figure 1.
Figure 1.
Targeted knock-in of Emerald GFP reporter in Zscan4 gene cluster reveals endogenous activity of Zscan4c locus in ES cells. (A) Schematic illustrations of the 7qA1 region of mouse chromosome 7, where multiple Zscan4 genes (Zscan4a∼f) and pseudogenes (Zscan4-ps1∼3) cluster (arrows: direction of transcription from each Zscan4 gene or pseudogene). The wild-type Zscan4c allele, Zscan4c-Emerald knock-in (Z4c-Emerald-KI) allele, and targeting vector are shown. Targeted replacement of Exon 2 and the following intron of the Zscan4c locus with Emerald GFP results in Z4c-Emerald-KI allele, in which Emerald GFP is under an endogenous Zscan4c promoter. Blue boxes represent exons. Black boxes represent Frt sites. Blue triangles represent lox P sites. Arrowheads indicate the targeting sites of guide RNAs for an hSpCas9-mediated DNA double-strand break. (B) FISH using an FITC-labeled probe that specifically detects the portion of Emerald GFP-Neo in the knock-in allele showed a single dot or a pair of single dots (two juxtaposed signals derived from sister chromatids) before or after the S phase, respectively, indicating that one allele of Zscan4c was replaced by the knock-in vector in the Z4c-Emerald-KI ES clone. Scale bar, 1 μm. (C) Emerald+ cells were infrequently observed in the Z4c-Emerald-KI ES colonies. Left: bright-field image; right: GFP fluorescent image. Scale bars, 50 μm. (D) Fluorescence-activated cell sorting (FACS) analyses of Emerald+ (EM+) ES population in the parental TA1 ES cells (left, negative control), the Z4c-Emerald-KI ES cell clone no. 27 (middle), and MC1 pZ4c-Emerald-Tg ES cells (right) in the conventional (FBS+ LIF) ES culture condition. Note that the overall scatter plot in MC1 pZ4c-Emerald-Tg ES cells shifts toward the right, indicating that the overall Emerald intensity is higher compared to that in Z4c-Emerald-KI ES cells. (E) Zscan4c-Emerald GFP knock-in ES cells were immunostained as indicated using rabbit anti-Zscan4. Arrows: GFP+/Zscan4+ cell; arrowheads: GFP−/Zscan4+ cell; asterisks GFP weakly+/Zscan4 weakly+ cell. Scale bar, 10 μm. Zscan4/GFP-immunostained ES cells were quantified, showing that 1.03% of the total ES cells were Zscan4+ (left graph) and 35.3% of Zscan4-immunopositive cells were GFP+/Zscan4+ (right graph). None of the GFP+/Zscan4-cells was observed. (F) ES cells were subjected to in situ hybridization of bulk Zscan4 mRNA (ISH, red) and immunostaining by rabbit anti-Zscan4 antibody (IF, green), demonstrating that anti-Zscan4 immunopositivity correlates with the expression of bulk Zscan4 mRNA. Note that bulk Zscan4 mRNA is detected over cytosol, while the Zscan4 protein is immunostained in nuclei. Scale bar, 10 μm.
Figure 2.
Figure 2.
The Zscan4c-Emerald GFP reporter knock-in mouse revealed endogenous activities of Zscan4 loci in the mouse preimplantation embryo. (A) GFP fluorescent images of two-cell stage embryos collected from a heterozygous Z4c-Emerald-KI female mouse crossed with a wild-type male at 46 h (upper left) and 51 h (upper right) post hCG. The white arrowhead indicates Emerald+ two-cell embryo. The number of Emerald+ two-cell embryos is summarized in the table (lower). Scale bar, 200 μm. (B) The late two-cell stage embryos at post 51 h hCG were immunostained with anti-GFP. Z4c-Emerald KI two-cell embryo showed GFP staining, while polar bodies or wild-type two-cell embryos did not. Scale bar, 50 μm. (C) Schematic illustrations of the wild-type Zscan4c allele (+), Zscan4c-Emerald GFP-Neo knock-in allele (Neo), and Zscan4c-Emerald GFP-NeoΔ knock-in allele (NeoΔ) are shown (upper). The NeoΔ allele knock-in mouse line was generated by crossing a Neo knock-in mouse with a transgenic mouse expressing Flippase to delete pGK-Neo-polyA franked by Frt sequences. Arrows: PCR primes. Due to the highly identical nucleotide nature of Zscan4 multigene loci, wild-type Zscan4c locus cannot be distinguished from other Zscan4 loci by PCR genotyping. To facilitate identification of homozygous Zscan4c-Emerald knock-in siblings, Neo/+ mouse was crossed with NeoΔ/+, producing homozygous Neo/NeoΔ, which was identified by PCR (lower left). Homozygous Neo/NeoΔ siblings, in which functional Zscan4c allele is disrupted, are viable (lower right).
Figure 3.
Figure 3.
The Zscan4c-Emerald GFP reporter knock-in locus is activated by retinoic acid in ES cells. (A) GFP fluorescence images of the Z4c-Emerald-KI ES cells in the presence of VPA (0.5 μM) or RA (1 μM, 10 μM) and of the MC1 ES cells with Z4c-GFP-IRES-Puro transgene in the presence of RA (10 μM) and puromycin. Scale bar, 200 μm. (B) FACS analyses of the GFP+ population in the Z4c-Emerald-KI ES cells and MC1 Z4c-GFP-IRES-Puro Tg ES cells as indicated in (A). Note that the overall scatter plot in Z4c-GFP-IRES-Puro Tg ES cells, which acquire puromycin resistance only when GFP is expressed, shifts toward the right, indicating that the overall GFP intensity of the survived cells selected against puromycin is higher compared to that in Z4c-Emerald-KI ES cells.
Figure 4.
Figure 4.
Mass spectrometry analysis revealed the expression of endogenous Zscan4 proteins and their associated factors in Z4c-Emerald knock-in ES cells. (A) Emerald+ mouse ES cells after RA treatment for 48 h were separated into chromatin bound and unbound fractions. Immunoprecipitation (IP) was done by control IgG and rabbit anti-Zscan4 antibody from chromatin-bound and chromatin-unbound fractions. Western blotting showed that endogenous Zscan4 protein was exclusively extracted from the chromatin-bound fraction. (B) Silver-stained gel showing immunoprecipitates (IP) by control IgG and rabbit anti-Zscan4 antibody from the chromatin fraction of Emerald+ ES cells, enriched by FACS sorting after RA treatment. M: molecular weight marker. Arrowhead: Zscan4. (C) LC-MS/MS analysis of immunoprecipitated endogenous Zscan4 showing unique MS spectrums specifically assigned to peptide sequences derived from Zscan4c, Zscan4d, or Zscan4f. (D) Western blotting of immunoprecipitated endogenous Zscan4 probed with rabbit anti-Zscan4 antibody. Arrowhead: Zscan4. Asterisk: IgG heavy chain. (E) Whole-cell extracts of RA-treated Z4c-Emerald KI ES cells after administration of Zscan4 or negative control siRNA were probed with antibodies as indicated. Note that specificity of the immunoreactive band detected by rabbit anti-Zscan4 antibody was confirmed by siRNA against Zscan4 genes. (F) List of proteins identified in the Zscan4 immunoprecipitates by LC-MS/MS analysis. Spectral counts (SpC) of the identified peptide are indicated. Highlighted in red are the Zscan4-associated factors identified in the previous MS analysis of Zscan4c-FLAG immunoprecipitates after overexpression (Akiyama et al. 2015).

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