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Generation of WT1-tdTomato knock-in cynomolgus monkey embryonic stem cell line, WT1-205 using CRISPR/CAS9-based gene

Ahjol Hyraht1, Fenglin Zhan1, Rongrong Guo1

  • 1State Key Laboratory of Primate Biomedical Research, Institute of Primate Translational Medicine, Kunming University of Science and Technology, Kunming, Yunnan 650500, China.

Stem Cell Research
|June 4, 2025
PubMed

Insights

Researchers created a WT1 (Wilms Tumor 1) reporter stem cell line in cynomolgus monkeys. This tool tracks cells developing into kidneys and gonads, advancing developmental biology research.

Area of Science:

  • Developmental Biology
  • Stem Cell Biology
  • Genetics

Background:

  • The Wilms Tumor 1 (WT1) gene is essential for the development of intermediate structures like the kidney, gonad, and adrenal cortex.
  • Understanding WT1 gene function is critical for studying organogenesis and developmental abnormalities.

Purpose of the Study:

  • To generate a WT1 reporter cynomolgus embryonic stem cell (cyESC) line for tracking WT1-expressing cells.
  • To validate the utility of this reporter line in studying intermediate mesodermal development.

Main Methods:

  • CRISPR/Cas9 technology was used to knock in a tdTomato reporter gene at the WT1 stop codon in cyESCs.
  • A PGK-Neo selection cassette (LSL) was included and subsequently excised by Cre recombinase.
  • In vitro differentiation protocols were employed to generate kidney- and gonadal-like cells.

Main Results:

  • tdTomato fluorescence was observed in differentiating cells, indicating successful WT1 gene targeting.
  • Co-localization of tdTomato with known markers (GATA4 for gonadal, PAX2 for kidney) confirmed the reporter's specificity.
  • WT1-positive cells expressed other relevant intermediate mesodermal genes.

Conclusions:

  • The generated WT1-tdTomato cyESC line is a valuable tool for investigating WT1-driven developmental processes in non-human primates.
  • This reporter line facilitates the study of intermediate mesodermal development and cell fate determination.
  • The findings contribute to a better understanding of primate embryogenesis and potential applications in regenerative medicine.

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