B6-hPCSK9 Mice

Catalog Number: I001179

Strain Name: C57BL/6NCya-Pcsk9tm1(hPCSK9)/Cya

Genetic Background: C57BL/6NCya

 

Strain Description

Proprotein convertase subtilisin/kexin 9 (PCSK9) is a serine protease primarily produced in the liver but expressed in other tissues, including the intestine, heart, and neurons. The N-terminal domain of the PCSK9 protein is responsible for protein localization and stability, while the C-terminal domain is responsible for protein enzymatic activity [1]. The Low-density lipoprotein receptor (LDLR) is a receptor that is responsible for clearing low-density lipoprotein cholesterol (LDL-C) from the blood. PCSK9 cleaves the intracellular domain of LDLR on the cell surface, causing it to detach from the cell membrane and be transported to the lysosome for degradation, promoting LDLR degradation, and increasing plasma LDL-C. Overexpression or gain-of-function mutations of the PCSK9 gene can lead to LDL-C accumulation by reducing LDLR levels. This can cause hypercholesterolemia, which increases the risk of cardiovascular diseases, such as atherosclerosis and coronary heart disease, and neurodegenerative diseases, such as Alzheimer's disease [2]. PCSK9 has become an important target for the development of lipid-lowering drugs. Several PCSK9-targeted antibodies or small nucleic acid drugs have been approved for marketing worldwide, including evolocumab from Amgen, alirocumab from Sanofi and Regeneron, and inclisiran from Novartis. These drugs primarily work by inhibiting PCSK9 activity or preventing PCSK9 protein from binding to LDLR, lowering LDL-C levels in the blood to treat hypercholesterolemia [3-4]. In addition, PCSK9 can promote tumor growth and development by regulating cell proliferation, migration, and invasion. It can also regulate the expression of inflammatory factors that contribute to inflammation. Therefore, targeting the expression of PCSK9 has been investigated in tumor immunotherapy and autoimmune disease therapy [5-6].

B6-hPCSK9 mice are a humanized model generated by gene editing technology to replace the mouse Pcsk9 gene with the human PCSK9 gene sequence. These mice express human PCSK9 protein and can be used for research on various metabolic diseases, neurodegenerative diseases, tumor development, autoimmune disease mechanisms, and for the preclinical pharmacological evaluation of PCSK9-targeted drugs.

Strain Strategy


Figure 1. Gene editing strategy of B6-hPCSK9 mice. The mouse Pcsk9 gene sequence, including ~35.5 kb upstream and ~7 kb downstream sequences, was replaced with the corresponding sequences in the human PCSK9 gene, including the UTR regions.

Applications

  • Development and screening of PCSK9-targeted therapies;
  • Preclinical pharmacological and efficacy evaluation of PCSK9-targeted therapies;
  • Research on metabolic diseases such as hypercholesterolemia, atherosclerosis, and coronary heart disease;
  • Research on neurodegenerative diseases such as stroke and Alzheimer's disease.

 

Validation Data

1. Expression of human PCSK9 gene and mouse Pcsk9 gene

Figure 2. Expression of human PCSK9 and mouse Pcsk9 genes in the liver and colon of 6-week-old homozygous B6-hPCSK9 and wild-type (WT) mice (n=4). RT-qPCR analysis revealed significant expression of the human PCSK9 gene in the liver and colon of homozygous B6-hPCSK9 mice, with no detectable expression of the mouse Pcsk9 gene. In wild-type mice, only the mouse Pcsk9 gene was expressed. Furthermore, the expression of the human PCSK9 gene was higher in the liver compared to the colon of B6-hPCSK9 mice. Notably, female B6-hPCSK9 mice exhibited higher expression levels of the human PCSK9 gene in both the liver and colon than their male counterparts. (ND: Not detected)

2. Expression of the mouse Ldlr gene

Figure 3. Expression of the mouse Ldlr gene in the liver of 6-week-old homozygous B6-hPCSK9 and wild-type (WT) mice (n=4). RT-qPCR results show that the expression level of the mouse Ldlr gene in the liver of B6-hPCSK9 mice is higher than that in wild-type mice.

3. Expression of human PCSK9 protein

a. Western Blots


Figure 4. Expression of human PCSK9 protein (hPCSK9) in the liver and colon tissues of wild-type (WT) and B6-hPCSK9 mice. Human-specific antibodies were employed in Western Blot analyses to detect the expression of human PCSK9 protein in the liver and colon of WT and B6-hPCSK9 mice. During synthesis, human PCSK9 initially forms a precursor protein, which undergoes autocatalytic cleavage to yield the mature protein subsequently secreted extracellularly. The antibody can detect both the precursor and mature forms of human PCSK9 protein. The results demonstrate that B6-hPCSK9 mice successfully express both forms of human PCSK9 protein.

*The antibody used is specific to the human PCSK9 protein. The bands observed in WT mice may be non-specific bands due to cross-reactivity.

b. ELISA


Figure 5. Expression of human PCSK9 protein in wild-type (WT) and B6-hPCSK9 mice serum. Human-specific antibodies were utilized in ELISA assays to detect the presence of human PCSK9 protein in the serum of WT and B6-hPCSK9 mice. The results revealed significant expression of human PCSK9 protein in the serum of B6-hPCSK9 mice, whereas no expression was detected in WT mice. (6 weeks old; homozygous; n=3; mice were not fasted prior to blood collection; data presented as mean ± SD)

4. Stable expression of human PCSK9 protein


Figure 6. ELISA of human PCSK9 protein in serum of B6-hPCSK9 mice at different age stages (n=5). The results showed that B6-hPCSK9 mice maintained stable expression of human PCSK9 protein for six consecutive weeks between 6 and 11 weeks of age. (Normal chow diet; mice were not fasted before blood collection; Bars represent mean ± SD)

5. Blood Biochemistry


Figure 7. Blood biochemical assay results of 11-week-old homozygous B6-hPCSK9 mice and wild-type (WT) mice (n=5). The results showed that the levels of T-CHO, HDL-C, LDL-C, and ALT were lower in B6-hPCSK9 mice than in WT mice, while the levels of TG and AST were not significantly different. (Mice were not fasted before blood collection; *p<0.05;**p<0.01;***p<0.001; Bars represent mean ± SEM)

* TG: Triglycerides; T-CHO: Total cholesterol; HDL-C: High-density lipoprotein cholesterol; LDL-C: Low-density lipoprotein cholesterol; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase.

 

References
[1]Melendez QM, Krishnaji ST, Wooten CJ, Lopez D. Hypercholesterolemia: The role of PCSK9. Arch Biochem Biophys. 2017 Jul 1;625-626:39-53.
[2]Seidah NG, Awan Z, Chrétien M, Mbikay M. PCSK9: a key modulator of cardiovascular health. Circ Res. 2014 Mar 14;114(6):1022-36.
[3]Pasta A, Cremonini AL, Pisciotta L, Buscaglia A, Porto I, Barra F, Ferrero S, Brunelli C, Rosa GM. PCSK9 inhibitors for treating hypercholesterolemia. Expert Opin Pharmacother. 2020 Feb;21(3):353-363.
[4]Sabatine MS. PCSK9 inhibitors: clinical evidence and implementation. Nat Rev Cardiol. 2019 Mar;16(3):155-165.
[5]Ding Z, Pothineni NVK, Goel A, Lüscher TF, Mehta JL. PCSK9 and inflammation: role of shear stress, pro-inflammatory cytokines, and LOX-1. Cardiovasc Res. 2020 Apr 1;116(5):908-915.
[6]Liu X, Bao X, Hu M, Chang H, Jiao M, Cheng J, Xie L, Huang Q, Li F, Li CY. Inhibition of PCSK9 potentiates immune checkpoint therapy for cancer. Nature. 2020 Dec;588(7839):693-698.