4s2 in liver

Author

Steve & Jose

Published

October 23, 2025

Mouse Models and Experimental Design

Animal Models

All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) and complied with relevant ethical guidelines.

ApoE4→ApoE2 Hepatocyte Switch Model

The primary experimental cohort consisted of ApoE4^flox/flox mice (JAX #034676), which enable Cre-mediated conversion of the ApoE4 allele to ApoE2. At 2 months of age, male and female mice received a single retro-orbital injection of either:

  • AAV8-TBG-EGFP-T2A-iCre (Cre group; Vector Biolabs, catalog #VB1725), or
  • AAV8-TBG-EGFP (GFP control; Vector Biolabs, catalog #VB1743).

The Thyroid hormone-binding globulin (TBG) promoter confers hepatocyte-specific expression, ensuring that recombination occurs exclusively in the liver. The bicistronic construct contains enhanced green fluorescent protein (EGFP) linked via the Thoseaasigna virus 2A (T2A) self-cleaving peptide to improved Cre recombinase (iCre), enabling stoichiometric (1:1) expression of EGFP and iCre from a single transcript. This design produces ApoE2 expression in hepatocytes, while maintaining ApoE4 expression in all extrahepatic tissues, including brain. Mice injected with the control AAV8-TBG-EGFP vector retain global ApoE4 expression.

Targeted-Replacement Controls

As reference isoform controls, age-matched 12-month-old male ApoE targeted-replacement (TR) mice expressing human ApoE2, ApoE3, or ApoE4 were included in Batch 2 only.

5XFAD Status

All TR controls and switch mice (Cre and GFP) in this batch were hemizygous for the 5XFAD transgene (5XFAD⁺/⁻), which carries human APP and PSEN1 mutations driven by the neuron-specific Thy1 promoter. The 5XFAD genotype permits assessment of ApoE isoform and hepatocyte-specific effects in the context of amyloidogenic background.

Sample Collection and batches

Animals were euthanized at 12 months of age, and liver and brain tissues were rapidly dissected, flash-frozen in liquid nitrogen, and stored at −80 °C until RNA extraction. In total, two sequencing batches were generated:

  • Batch 1: 40 samples (20 liver, 20 brain). Of these, 18 samples (9 liver and 9 brain) were confirmed to be correctly genotyped and retained for further analysis. Two samples (SAD_326 and MAD_271) were re-sequenced in Batch 2 to assess inter-batch reproducibility.

  • Batch 2: 44 new samples (22 liver, 22 brain) comprising Cre, GFP, and 5XFAD+/– TR controls (E2, E3, E4).

Thus, the final dataset included 62 samples across tissues and experimental groups.