Research Models

APOE3 Knock-In (JAX)

Synonyms: APOE3 KI, APOE*3 KI

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Species: Mouse
Genes: APOE
Modification: APOE: Knock-In
Disease Relevance: Alzheimer's Disease
Strain Name: B6.Cg-Apoeem2(APOE*)Adiuj/J
Genetic Background: C57BL/6J
Availability: The Jackson Laboratory, Stock # 029018; Live

Summary

The APOE3 knock-in model is one line in a set of three APOE knock-in lines created by The Jackson Laboratory (JAX). APOE2 and APOE4 mice are also available.

In this set of APOE knock-in mice, the coding sequence of the endogenous murine Apoe gene was replaced by one of the three major human APOE alleles (E2, E3, or E4), but expression of the gene remains under the control of mouse regulatory elements. The models created and distributed by The Jackson Laboratory are similar to APOE Targeted Replacement mice. However, there are no breeding restrictions on JAX mice—meaning that researchers can generate mice with heterozygous pairings of APOE alleles, and breeding strategies can be implemented to compare different genotypes in litter-matched mice.

Expression

A 2022 study compared JAX’s APOE3 and APOE4 knock-in mice to each other and to APOE Targeted Replacement mice (Sepulveda et al., 2022). Similar to previous findings in the APOE Targeted Replacement models, levels of APOE mRNA did not differ between JAX’s APOE3 and APOE4 knock-in mice, but less ApoE protein was measured in the cortices of APOE4 mice than APOE3 mice when homogenates were prepared using detergent-containing buffers.

Peripheral phenotypes

Preliminary characterization of the APOE4 strain—the first of the three knock-in lines made by Jackson—was reported at the 2018 AAIC meeting. At 4 and 14 months, levels of total cholesterol and LDL were decreased in the sera of male and female APOE4 knock-In mice, compared with C57BL/6J controls. A reduction in the levels of HDL was seen in males at 4 months and in both genders at 14 months. Levels of triglycerides and non-essential fatty acids were normal. While control mice gained weight, at least until 14 months of age, the body weights of APOE4 knock-in mice were stable between 4 and 14 months.

Subsequently, JAX scientists crossed APOE3 and APOE4 knock-in mice, then intercrossed the offspring to produce litter-matched E3/E3, E3/E4, and E4/E4 carriers. Mice were initially characterized at 2 and 4 months of age (Foley, Hewes et al., 2022), with a second report extending observations to 12 months (Foley, Diemler et al., 2022). APOE genotype did not influence body weight, unfasted glucose levels or the concentrations of total cholesterol, HDL, or LDL in the plasma of young (2- and 4-month-old) mice. However, there was an effect of sex, with males being heavier and having higher levels of plasma cholesterol, HDL, and glucose than females. Levels of plasma triglycerides, measured in 2-month-old mice, did not depend on APOE genotype or sex. At 12 months, APOE genotype did not affect overall weight, lean mass, fat mass, plasma total cholesterol, or plasma HDL.

Brain lipids

Neither APOE genotype nor sex influenced the concentrations of total cholesterol, HDL, or LDL in the brains of 2-month-old litter-matched E3/E3, E3/E4, and E4/E4 carriers (Foley, Hewes et al., 2022).

Synapses

As yet, there is little information about synaptic structure or function in the JAX APOE knock-in lines. However, one study found that spine density on basal dendrites of pyramidal neurons of the medial entorhinal cortex was lower in APOE4 homozygotes than APOE3 homozygotes, at 6 months of age (Sepulveda et al., 2022). The genotypes did not differ with regard to the density of spines on apical dendrites.

Gliosis

Information about glial phenotypes in JAX APOE knock-in mice is currently lacking. One study found that astroglial (GFAP immunoreactivity) and microglial (Iba1 immunoreactivity) markers were slightly, but not significantly, elevated in 8-month-old APOE4/4 females compared with age- and sex-matched APOE3/4 and APOE3/3 mice, although the authors of this study acknowledged that the study was underpowered to detect differences between genotypes (Onos et al., 2024).

Behavior

As mentioned above, preliminary characterization of JAX’s APOE4 knock-in mice was reported at the 2018 AAIC meeting. At 2 and 12 months of age, locomotor activity (distance traveled in an open field test) and motor coordination (latency to fall on rotarod test) were similar in APOE4 knock-in and C57BL/6J control mice, with both genotypes showing an age-dependent decline in these measures. Two- and 12-month-old APOE4 mice also performed similarly to control mice in a Y-maze test of working memory.

When tested at 6 months of age, APOE3 and APOE4 knock-in mice performed similarly in the Barnes maze, a test of spatial learning and memory, and in the open field and elevated zero maze, assays of activity, exploratory behavior, and anxiety (Sepulveda et al., 2022).

A study comparing aged (approximately 23 months) APOE3/3, APOE3/4, and APOE4/4 mice found that performance in the open field and novel object recognition (NOR) tests depended more upon sex than genotype (McLean et al., 2022). Females more strongly avoided the center of the open field than did males and spent more time moving and interacting with objects during the familiarization phase of the NOR test. There were no genotype- or sex-dependent differences in the metric used to evaluate animals’ ability to recognize a novel object when tested 24 hours after familiarization [discrimination index = (time spent with novel object - time spent with familiar object)/total time spent with both objects]. However, only female APOE4/4 mice appeared to recognize the novel object (i.e., discrimination index > 0).

Bioenergetics

The influence of APOE genotype on mitochondrial respiration and glycolysis has been studied using acute hippocampal slices from 3-month-old male APOE3 and APOE4 mice (Qi et al., 2021). Under these ex vivo conditions, E4 slices showed increased glucose metabolism and decreased fatty-acid metabolism, compared with E3 slices. The cellular basis of these differences was explored using cultured neurons and astrocytes from the brains of APOE3 and APOE4 knock-in mice, as mentioned below under “Applications of the model.”

Transcriptomics

In a 2022 study, APOE3 and APOE4 knock-in mice were crossed, then the offspring intercrossed to produce litter-matched E3/E3, E3/E4, and E4/E4 carriers (Foley, Hewes et al., 2022). The cortical transcriptomes of the E3/E4 heterozygotes and E4/E4 homozygotes were compared with E3/E3 homozygotes. Differentially expressed genes (DEGs) in 2-month-old E3/E4 heterozygotes included those involved in hormone regulation and insulin signaling, while genes involved in angiogenesis and cell adhesion were among those differentially expressed in E4/E4 homozygotes. Interestingly, the majority of DEGs uniquely expressed in E3/E4 brains have been found to be expressed in endothelial cells and astrocytes, while those uniquely expressed in E4/E4 brains are neuronally expressed. At 4 months, DEGs in E4 carriers—both homozygotes and heterozygotes—were enriched for Gene Ontology (GO) terms “MHC protein complex” and “BMP receptor binding.” DEGs unique to E4/4 homozygotes were enriched for GO terms “branching morphogenesis of an epithelial tube,” “chemotaxis,” and “taxis.” DEGs unique to APOE3/4 heterozygotes were enriched for “extracellular matrix,” “collagen-containing extracellular matrix,” and “blood coagulation,” leading the study’s authors to speculate that the cerebrovasculature may be damaged in APOE3/4 mice.

Another 2022 study measured the relative expression of neuroinflammatory markers in the cortices of 8-month-old JAX APOE3 and APOE4 knock-in mice and APOE3 and APOE4 Targeted Replacement mice (Sepulveda et al., 2022). Comparison of the two sets of models revealed some differences: While the level of Serpina3n was much higher in APOE4 Targeted Replacement mice compared with APOE3 Targeted Replacement mice, transcript levels did not differ between JAX APOE3 and APOE4 knock-ins. By contrast, higher levels of Il3 mRNA were found in Jackson APOE4 knock-ins than in APOE3 mice, but transcript levels did not differ between APOE3 and APOE4 Targeted Replacement mice.  Levels of Tnf and Il6 were similar in E3 and E4 carriers, in both JAX knock-ins and Targeted Replacement models.

Neurovascular coupling

Neurovascular coupling is the process by which cerebral blood flow is adjusted to match the energy needs imposed by neural activity. A 2024 study examined age-dependent changes in neurovascular coupling as a function of genotype in APOE knock-in mice (Onos et al., 2024). PET imaging was used to measure the brain distributions of 18F-FDG (fluordeoxyglucose) and 64Cu-PTSM (pyruvaldehyde bis(N4-methyl)thiosemicarbazone) as proxies for neural activity and cerebral perfusion, respectively.

The data generated by this study were complex with regards to the effects of age, sex, and genotype on regional perfusion and glucose uptake. Nonetheless, some conclusions are summarized here. The study compared differences between age groups (8 or 12 months vs. 4 months) in glucose uptake and perfusion within each genotype. The neurovascular response to aging was considered “coupled” if changes in both glucose uptake and perfusion were in the same direction (i.e., both increased or both decreased in a given region). Conversely, the response was “uncoupled” if either perfusion increased while glucose uptake decreased (“Type 1” uncoupling) or perfusion decreased while glucose uptake increased (“Type 2” uncoupling). For APOE3/3 mice, the majority of brain regions showed a coupled response to aging between 4 and 8 months; between 4 and 12 months, both males and females showed some uncoupling in a few, non-overlapping regions. Uncoupled responses in APOE4/4 mice were more extensive and sex differences were more pronounced. Between 4 and 8 months, Type 1 uncoupling was observed in 23 of 27 regions in female mice and 11 of 27 regions in male mice. Between 4 and 12 months, females continued to show Type 1 uncoupling (20/28 regions), but males mainly showed Type 2 uncoupling (15/28 regions). Heterozygous carriers of the E4 allele also showed sex differences—between 4 and 8 months APOE3/4 females showed an uncoupled response (Type 2) in many more regions than did males.

Applications of the model

Following is a short survey of some of the early studies employing JAX APOE knock-in mice.

Effect of APOE genotype on responses to exercise. One study compared the cortical transcriptomes of 4-month-old APOE3/3 and APOE4/4 mice that had been provided access to a running wheel for 3 months with the transcriptomes of sedentary animals.  A genotype x exercise interaction was not seen (Foley, Hewes et al., 2022). A second study examined the effects of 11 months of voluntary exercise, beginning at 1 month of age, in litter-matched E3/E3, E3/E4, and E4/E4 carriers, (Foley, Diemler et al., 2022). APOE genotype interacted with running to affect multiple phenotypes—including body mass, energy expenditure, and cortical and hippocampal transcriptomes.

Effect of APOE genotype on HFD-induced insulin resistance. This study examined the effect of APOE genotype on the development of insulin resistance induced by a high-fat diet (Rai et al., 2023). Beginning at 2 months of age, male APOE3 and APOE4 mice were randomly assigned to regular or high-fat diets. Animals were maintained on these diets for 1, 3, or 6 months (or until 3, 5, or 8 months of age). Compared with mice fed a regular diet, E4 carriers on the high-fat diet had increased body weights by 3 months of age, increased levels of plasma insulin by 5 months of age, and increased levels of blood glucose by 8 months. Neither plasma insulin nor blood glucose were influenced by diet in APOE3/3 mice up to 8 months of age, although E3 carriers fed a high-fat diet were heavier than those fed a regular diet at 8 months.

Bioenergetics of neurons and astrocytes. This study compared aspects of fatty acid metabolism in neurons and astrocytes cultured from the brains of fetal and 3-month-old APOE knock-in mice, respectively (Qi et al., 2021). Compared with neurons from APOE3 mice, APOE4 neurons less efficiently sequestered fatty acids in lipid droplets. Fatty acid oxidation was impaired in E4 astrocytes, compared with E3.

Genetic risk plus inflammatory stimulus. JAX’s APOE4 knock-in mice were used to study the effects of systemic inflammation on behavior, dendritic structure, and numbers of glial cells (Ganesan et al.,  2024). Beginning at 2 months of age, animals received weekly intraperitoneal injections of LPS for 4 months. LPS treatment decreased activity in the open field and Y-maze and reduced the numbers of dendritic spines on apical dendrites of hippocampal CA1 pyramidal neurons but did not affect spatial learning and memory in the Barnes maze or numbers of astrocytes and microglia in the hippocampus.

Response to treatment in a model of non-traumatic spinal cord injury. JAX’s APOE3 and APOE4 knock-in mice were first subjected to a surgical procedure leading to progressive compression of the spinal cord, in order to  model degenerative cervical myelopathy. Six weeks later, surgical decompression was performed, and mice were evaluated one day to 5 weeks after decompression surgery. Compared with APOE3 mice, APOE4 mice showed delayed functional recovery (gait, forelimb grip strength, and hindlimb mechanical sensitivity), increased levels of circulating cytokines (TNF-α, IL-6, CCL3, and CXCL9), and decreased gray matter area accompanied by increased microgliosis and astrogliosis at the site of injury (Desimone et al., 2021).

Effect of ibuprofen on dendritic spine density. Rebeck and colleagues found lower dendritic spine densities in the entorhinal cortices of 6-month-old JAX’s APOE4 knock-in mice compared with APOE3 mice (Sepulveda et al., 2022). Short-term treatment with the non-steroidal anti-inflammatory drug ibuprofen increased the number of dendritic spines in the E4 mice, bringing the levels up to those of E3 mice (ibuprofen did not further increase the spine density of E3 mice). These findings confirm earlier observations by Rebeck’s group in APOE Targeted Replacement mice (DiBattista et al., 2016).

Effect of APOE genotype on microglial activity. APOE knock-in mice were crossed with mice that express green fluorescent protein driven by the CX3CR1 promoter to visualize living microglia. Ex vivo imaging of brain slices revealed that the processes of E4 microglia moved more slowly during baseline surveillance of their environment and in response to application of ATP (injury signal) or Aβ than did the processes of E3 microglia (Sepulveda et al., 2024).

Modification Details

In these models, exons 2, 3 and most of exon 4 of the mouse Apoe gene were replaced by human APOE gene sequence including exons 2, 3, 4, and part of the 3' UTR—resulting in substitution of  the mouse coding sequence with the human coding sequence and retention of the mouse regulatory sequence and the non-coding exon one. The APOE4 knock-in model was created first, through homologous recombination in mouse embryonic stem cells, using a targeting vector containing 1.5 kb of human APOE gene sequence. Subsequently, the APOE2 and APOE3 knock-in models were created through CRISPR-Cas9 editing of the humanized APOE gene in APOE4-knock-in zygotes.

Last Updated: 28 Feb 2025

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References

Research Models Citations

  1. APOE2 Knock-In (JAX)
  2. APOE4 Knock-In (JAX)
  3. APOE3 Targeted Replacement

Paper Citations

  1. Sepulveda J, Luo N, Nelson M, Ng CA, Rebeck GW. Independent APOE4 knock-in mouse models display reduced brain APOE protein, altered neuroinflammation, and simplification of dendritic spines. J Neurochem. 2022 Nov;163(3):247-259. Epub 2022 Jul 29 PubMed.
  2. Foley KE, Hewes AA, Garceau DT, Kotredes KP, Carter GW, Sasner M, Howell GR. The APOE ε3/ε4 Genotype Drives Distinct Gene Signatures in the Cortex of Young Mice. Front Aging Neurosci. 2022;14:838436. Epub 2022 Mar 16 PubMed.
  3. Foley KE, Diemler CA, Hewes AA, Garceau DT, Sasner M, Howell GR. APOE ε4 and exercise interact in a sex-specific manner to modulate dementia risk factors. Alzheimers Dement (N Y). 2022;8(1):e12308. Epub 2022 Jun 29 PubMed.
  4. Onos KD, Lin PB, Pandey RS, Persohn SA, Burton CP, Miner EW, Eldridge K, Kanyinda JN, Foley KE, Carter GW, Howell GR, Territo PR. Assessment of neurovascular uncoupling: APOE status is a key driver of early metabolic and vascular dysfunction. Alzheimers Dement. 2024 Jul;20(7):4951-4969. Epub 2024 May 7 PubMed.
  5. McLean JW, Bhattrai A, Vitali F, Raikes AC, Wiegand JL, Brinton RD. Contributions of sex and genotype to exploratory behavior differences in an aged humanized APOE mouse model of late-onset Alzheimer's disease. Learn Mem. 2022 Sep;29(9):321-331. Print 2022 Sep PubMed.
  6. Qi G, Mi Y, Shi X, Gu H, Brinton RD, Yin F. ApoE4 Impairs Neuron-Astrocyte Coupling of Fatty Acid Metabolism. Cell Rep. 2021 Jan 5;34(1):108572. PubMed.
  7. Rai A, Ojiakor OA, Rylett RJ. Detection of early Alzheimer's disease-like molecular alterations in a mouse model expressing human ApoE4. J Neurochem. 2023 Aug;166(3):572-587. Epub 2023 Jul 6 PubMed.
  8. Ganesan K, Rentsch P, Langdon A, Milham LT, Vissel B. Modeling sporadic Alzheimer's disease in mice by combining Apolipoprotein E4 risk gene with environmental risk factors. Front Aging Neurosci. 2024;16:1357405. Epub 2024 Feb 27 PubMed.
  9. Desimone A, Hong J, Brockie ST, Yu W, Laliberte AM, Fehlings MG. The influence of ApoE4 on the clinical outcomes and pathophysiology of degenerative cervical myelopathy. JCI Insight. 2021 Aug 9;6(15) PubMed.
  10. DiBattista AM, Dumanis SB, Newman J, Rebeck GW. Identification and modification of amyloid-independent phenotypes of APOE4 mice. Exp Neurol. 2016 Jun;280:97-105. Epub 2016 Apr 14 PubMed.
  11. Sepulveda J, Kim JY, Binder J, Vicini S, Rebeck GW. APOE4 genotype and aging impair injury-induced microglial behavior in brain slices, including toward Aβ, through P2RY12. Mol Neurodegener. 2024 Mar 11;19(1):24. PubMed.

External Citations

  1. reported
  2. The Jackson Laboratory, Stock # 029018

Further Reading

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