Expression analysis of the endogenous Zscan4 locus and its coding proteins in mouse ES cells and preimplantation embryos

In Vitro Cell Dev Biol Anim. 2017 Feb;53(2):179-190. doi: 10.1007/s11626-016-0097-y. Epub 2016 Oct 3.


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.

MeSH terms

  • Animals
  • Blastocyst / drug effects
  • Blastocyst / metabolism*
  • Chromosomal Proteins, Non-Histone / genetics*
  • Chromosomal Proteins, Non-Histone / metabolism
  • Female
  • Gene Expression Regulation, Developmental* / drug effects
  • Gene Knock-In Techniques
  • Gene Targeting
  • Genes, Reporter
  • Genetic Loci*
  • Green Fluorescent Proteins / metabolism
  • Mass Spectrometry
  • Mice
  • Mice, Inbred C57BL
  • Mouse Embryonic Stem Cells / cytology
  • Mouse Embryonic Stem Cells / drug effects
  • Mouse Embryonic Stem Cells / metabolism*
  • Open Reading Frames / genetics*
  • Promoter Regions, Genetic
  • Transcription Factors / genetics*
  • Transcription Factors / metabolism
  • Tretinoin / pharmacology


  • Chromosomal Proteins, Non-Histone
  • Transcription Factors
  • Green Fluorescent Proteins
  • Tretinoin