연구자들이 COVID-19 병리에서의 ACE2의 의미를 연구함에 따라 ACE2의 전반적인 메커니즘을 탐색하기 위한 다양한 동물 모델이 필요하게 됐다. H11 knock-in(KI), ROSA26 Conditions knock-in(cKI) 또는 내원성 대체 등 다양한 방법의 인간화 ACE2(hACE2) 모델을 개발할 수 있으며, 이러한 방법들은 서로 다른 단백질을 생성할 수 있다. 대부분의 동물모델에서 수행되는 ACE2 연구의 초기 단계를 고려하면 인간화 모델 생성 방법의 명확한 이점을 확인할 증거가 충분하지 않다.
이러한 도전에도 불구하고 다양한 동물 모델을 사용하여 COVID-19 감염의 발병 시 ACE2 의 기능을 설명할 수 있다. 우리의 K18-hACE2 마우스는 SARS-CoV-2 S2' 조각을 생성하는 데 도움을 주며, 바이러스와 수용체 간의 인식과 막 융합에서의 S2' 조각의 역할을 밝혀냈다. 이 주제는 "K18 promoter-kozaka-human ACE2 CDS-P2A-CreERT2-rBGpA" 카세트(cassette)를 마우스 H11 로커스(locus)에 삽입한다.
최근 PNAS 문장 K18-hACE2 마우스를 사용하여 ACE2 의 spike engagement이 과녁 세포에서 S2 조각의 생성을 유도하는 것으로 나타났는데, 이는spike-mediated 막 융합과 결합된 핵심 단백질 가수 분해 사건이다. Arginine(R) 815의S2' 절단 부위(cleavage site)는Arginine 685 의S2'절단 부위를 유인하는 대신 다양한 세포주와 인간화 ACE2 (hACE2) knock-in마우스에서 분리된 원대세포에서 유인하는 것으로 후속 융합체의 형성과 감염을 막을 수 있다.이 연구는 숙주 수용체 접촉의 중요한 역할과 spike의 핵심 잔여물(residue)가 단백질 가수분해에 의한 활성화에 미치는 역할을 강조하고 SARS-CoV-2.1 감염 숙주 세포의 과녁 메커니즘을 밝혀냈다.
| 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 |
Cyagen은 이들 모델 외에도 DPP4와 APN 유전자 표적을 포함한 다른 COVID-19 모델을 우선적으로 연구하고 있다.원하는 모델을 록인하는 방법에 대한 자세한 내용은 지금 바로 문의하십시오.
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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