As researchers investigate the implications of ACE2 in COVID-19 pathology, different animal models are needed to explore the mechanisms of ACE2 in its entirety. There are multiple approaches to developing humanized ACE2 (hACE2) models, including H11 knockin (KI), ROSA26 conditional KI (cKI), or endogenous replacement, which yield different proteins. Given the early stages of most ACE2 research performed in animal models, there has yet to be sufficient evidence to determine the explicit advantages of either humanized model generation method.
Despite these challenges, using a variety of animal models can serve to elucidate the function of ACE2 in onset of COVID-19 infection. Our K18-hACE2 Mice help generate the SARS-CoV-2 S2′ fragments, and have revealed the role of the S2’fragment in recognition and membrane fusion between virus and receptor. In this project, the "K18 promoter-Kozak-human ACE2 CDS-P2A-CreERT2-rBGpA" cassette is inserted into the mouse H11 locus.
A recent PNAS article used K18-hACE2 mice to show that the spike engagement of ACE2 primed the generation of S2′ fragments in target cells, a key proteolytic event coupled with spike-mediated membrane fusion. Mutagenesis of an S2′ cleavage site at the arginine (R) 815, but not an S2 cleavage site at arginine 685, was sufficient to prevent subsequent syncytia formation and infection in a variety of cell lines and primary cells isolated from humanized ACE2 (hACE2) knock-in mice. The study highlights an essential role for host receptor engagement and the key residue of spike for proteolytic activation, and uncovers a targetable mechanism for host cell infection by SARS-CoV-2.1
| ACE2 Catalog Models | Backgrounds | Model ordering |
|---|---|---|
| Knockout (KO) | C57BL/6J | |
| BALB/c | ||
| Humanized Mice (ROSA26 cKI) | C57BL/6J | |
| BALB/c | ||
| C57BL/6N | ||
| Humanized Mice (H11 Locus KI) | C57BL/6J | |
| Humanized Mice (Endogenous Replacement) | C57BL/6J | |
| BALB/c | ||
| C57BL/6N |
In addition to the above-listed models, Cyagen is also prioritizing inquiries on other COVID-19 models, including DPP4 and APN gene targets. Contact us now for more information about how to lock-in your desired models.
Reference:
Yu S, Zheng X, Zhou B, Li J, Chen M, Deng R, Wong G, Lavillette D, Meng G. SARS-CoV-2 spike
engagement of ACE2 primes S2' site cleavage and fusion initiation. Proc Natl Acad Sci U S A.
2022
Jan 4;119(1):e2111199119.
doi: 10.1073/pnas.2111199119. PMID: 34930824; PMCID: PMC8740742.
Genetic Overview
1. C57BL/6J ACE2 Knockout Mice
● Model Generation
Model Name: ACE2-KO
Strain Background: C57BL/6J
● IHC-P Validation:
After the model was established, different tissues of the mice were fixed in formaldehyde, embedded in paraffin, and evaluated by immunohistochemistry experiments. The negative results reported in the intestines, lungs, liver, brain, trachea, kidney, stomach, and heart confirmed that no measurable ACE2 expression occurred in knockout (KO) mice – indicating successful KO model generation.
● C57BL/6J Negative Control:
Tissues of C57BL/6J negative control mice were fixed in formaldehyde, embedded in paraffin, and evaluated by immunohistochemistry experiments. The result reported negative in the intestines, lungs, liver, brain, trachea, kidney, stomach, and heart.
1. C57BL/6J ACE2 Knockin Mouse (H11 Locus)
● Model Generation:
Model Name: H11-ACE2-KI
Strain Background: C57BL/6J
Construction Strategy: The human signal peptide, extracellular domain, transmembrane
domain, and intracellular domain were inserted into the mouse H11 locus.
● IHC-P Validation:
After the model was established, different tissues of the mice were fixed in formaldehyde, embedded in paraffin, and evaluated by immunohistochemistry experiments. The results showed different levels of ACE2 expression in the intestine, lung, liver, brain, trachea, kidney, stomach, and heart, while K18 promoter still has nonspecific expression, which allows selection according to different experimental needs.
● C57BL/6J Negative Control:
Tissues of C57BL/6J blank control mice were fixed in formaldehyde, embedded in paraffin, and evaluated by immunohistochemistry experiments. The result reported negative in the intestines, lungs, liver, brain, trachea, kidney, stomach, and heart.
1. C57BL/6J Humanized ACE2 (hACE2) Mice
● Model Generation:
Model Name: ACE2-KI
Strain Background: C57BL/6J
Construction Strategy: Retain the mouse signal peptide and endogenous PolyA; replace
the
extracellular domain, transmembrane domain, and intracellular domain of mouse Ace2
with
that
of human ACE2.
● qPCR Validation:
After the model was established, different tissues of the mice were taken for qPCR verification. The results show hACE2 was expressed in the small and large intestine, kidney, lung, and trachea, which means that the model can successfully express human ACE2 gene transcripts and the humanized model verification is successful at the genetic level.
● IHC-P Validation:
After the model was established, different tissues of the mice were fixed in formaldehyde, embedded in paraffin, and immunohistochemistry experiments were performed. The results showed hACE2 protein was expressed in the intestines, kidneys, lungs, and trachea - which further confirmed that the humanized model can successfully express human ACE2 protein, and the model verification is successful.
● C57BL/6J Negative Control:
Tissues of C57BL/6J blank control mice were fixed in formaldehyde, embedded in paraffin, and evaluated by immunohistochemistry experiments. The result reported negative in the intestines, lungs, liver, brain, trachea, kidney, stomach, and heart.
● WB Verification - Expression variations across organs:
The six tissues of mice including the large intestine, small intestine, kidney, brain, trachea, and lung were tested separately; the theoretical band size of human ACE2 is 92 KD - the protein may be too large due to glycosylation modification in the protein.
Conclusions:
1. In the brain and lung tissues, all target clones have no detectable bands that are significantly different from the wild type;
2. In kidney tissue, the theoretical band size is 92 KD, while the actual band size is 120 KD.
3. In the large intestine tissue, the theoretical band size is 92 KD, while the actual band size is 130 KD.
4. In tracheal tissue, the theoretical band size is 92 KD, while the actual band size is 120 KD.
5. In the small intestine tissue, the theoretical band size is 92 KD, while the actual band size is 130 KD.
The expression of ACE2 protein in humanized ACE2 (hACE2) mice varies greatly among individuals and different tissues.
2. BALB/c Humanized ACE2 (hACE2) Mice
● Model Generation:
Model Name: ACE2-KI Strain Background: BALB/c Construction Strategy: Retain the mouse signal peptide and endogenous PolyA, replace the extracellular domain, transmembrane domain, and intracellular domain of mouse Ace2 with that of human ACE2.
● IHC-P Validation:
After the model was established, different tissues of the mice were fixed in formaldehyde, embedded in paraffin, and evaluated by immunohistochemistry experiments. The results showed hACE2 was expressed in the intestines, lungs, liver, brain, trachea, kidney, stomach, and heart - which further confirmed that the humanized model can successfully express human ACE2 protein, and the model verification is successful.
● BALB/c Negative Control:
Tissues of BALB/c blank control mice were fixed in formaldehyde, embedded in paraffin, and evaluated by immunohistochemistry experiments. The result reported negative in the intestines, lungs, liver, brain, trachea, kidney, stomach, and heart.
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