Two-Step Algorithmic Selection of Interspecies Sequence-Mismatch-Based Housekeeping Genes for Precise Gene-Expression...

Area of Science:

• Biomedical Research
• Immunology
• Genetics

Background:

• Accurate gene expression normalization is critical for humanized mouse models.
• Selecting appropriate housekeeping genes (HGs) for human cells in mice is challenging due to interspecies differences.

Purpose of the Study:

• To identify and validate stable, tissue-specific housekeeping genes for gene expression analysis in human peripheral blood mononuclear cell (hPBMC)-humanized NSG mice.
• To develop a robust algorithm for HG selection in humanized mouse models.

Main Methods:

• Evaluated 10 candidate HGs for expression stability in human PBMCs using five algorithms.
• Identified genes with significant interspecies variation between human and mouse cell lines.
• Performed in vivo tissue-specific expression analysis in hPBMC-humanized NSG mice.

Main Results:

• Developed a two-step algorithm prioritizing interspecies differences and expression stability.
• Recommended tissue-specific HGs: ACTB (heart), EEF2 (liver, spleen), SDHA (lungs, kidneys), TFRC (hPBMCs).
• EEF2 and SDHA were suitable for liver; EEF2 showed superior performance for cytokine mRNA analysis.

Conclusions:

• Species- and tissue-specific patterns are crucial for HG selection in humanized mouse models.
• The recommended HGs enhance accuracy and reliability of gene expression analysis.
• Findings improve the utility of humanized mouse models for human disease research.