ACE2 마우스 모델 - ACE2 KO, KI, Humanized Mice

Project Type	Background Strain	Order
ACE2 Humanized Model	C57BL/6J
C57BL/6N
BALB/c
ACE2 Knock-In Model (H11 Locus)	C57BL/6J
ACE2 Conditional Knock-In Model (Rosa26 Locus)	C57BL/6J
BALB/c
C57BL/6N
ACE2 Knock-Out (KO) Model	C57BL/6J
BALB/c

1. C57BL/6J ACE2 Humanized Mice

● qPCR Validation:

The results:

a. 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 with that of human.

b. After the model was established, different tissues of the mice were taken for qPCR verification. The results show ACE2 was expressed in the small and large intestine, kidney, lung, and trachea, which means that the humanized model can successfully express human ACE2 protein, and the model verification is successful.

2. C57BL/6J ACE2 Humanized Mice

● IHC-P Validation:

The results:

a. 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 with that of human.

b. After the model was established, different tissues of the mice were fixed in formaldehyde, embedded in paraffin, and immunohistochemical experiments were performed. The results showed ACE2 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:

The results:

a. Mouse Model: C57BL/6J background, negative control.

b. Tissues of C57BL/6J blank control mice were fixed in formaldehyde, embedded in paraffin, and evaluated by immunohistochemical experiments. The result reported negative in the intestines, lungs, liver, brain, trachea, kidney, stomach, and heart.

3. C57BL/6J ACE2 Humanized Mice

● WB Verification - Expression variations across organs:

The results:

The six tissues of mice including the large intestine, small intestine, kidney, brain, trachea, and lung were tested separately; the theoretical band size of ACE2 is 92KD - 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 92KD, while the actual band size is 120KD.
3. In the large intestine tissue, the theoretical band size is 92KD, while the actual band size is 130KD.
4. In tracheal tissue, theoretical band size is 92KD, while the actual band size is 120KD.
5. In the small intestine tissue, the theoretical band size is 92KD, while the actual band size is 130KD.

The expression of ACE2 in humanized ACE2 mice varies greatly among individuals and different tissues.

1. BALB/c ACE2 Humanized Mice

● IHC-P Validation:

The results:

a. 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 with that of human.

b. After the model was established, different tissues of the mice were fixed in formaldehyde, embedded in paraffin, and evaluated by immunohistochemical experiments. The results showed ACE2 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:

The results:

a. Mouse Model: BALB/c background, negative control.

b. Tissues of BALB/c blank control mice were fixed in formaldehyde, embedded in paraffin, and evaluated by immunohistochemical experiments. The result reported negative in the intestines, lungs, liver, brain, trachea, kidney, stomach, and heart.

1. C57BL/6J ACE2 Knockin Mouse (H11 Locus)

● IHC-P Validation:

The results:

a. 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.

b. After the model was established, different tissues of the mice were fixed in formaldehyde, embedded in paraffin, and evaluated by immunohistochemical 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 can be selected according to different experimental needs.

● C57BL/6J Negative Control:

The results:

a. Mouse Model: C57BL/6J background, negative control.

b. Tissues of C57BL/6J blank control mice were fixed in formaldehyde, embedded in paraffin, and evaluated by immunohistochemical experiments. The result reported negative in the intestines, lungs, liver, brain, trachea, kidney, stomach, and heart.

1. C57BL/6J ACE2 Knockout Mice

● IHC-P Validation:

The results:

a. Model Generation:

Model Name: ACE2-KO

Strain Background: C57BL/6J

b. After the model was established, different tissues of the mice were fixed in formaldehyde, embedded in paraffin, and evaluated by immunohistochemical 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:

The results:

a. Mouse Model: C57BL/6J background, negative control.

b. Tissues of C57BL/6J blank control mice were fixed in formaldehyde, embedded in paraffin, and evaluated by immunohistochemical experiments. The result reported negative in the intestines, lungs, liver, brain, trachea, kidney, stomach, and heart.