Name: huPCSK9 Mouse
Brand: 싸이아젠(Cyagen)
SKU: C001617

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huPCSK9 Mouse

제품명

huPCSK9 Mouse

제품 ID

C001617

품종 계통

C57BL/6NCya-Pcsk9^tm1(hPCSK9)/Cya

Backgroud

C57BL/6NCya

상태

이 마우스 계통을 논문에서 사용할 경우, “huPCSK9 Mouse (카탈로그 번호 C001617)은 Cyagen에서 구입하였습니다.”라고 명시해 주시기 바랍니다.

HUGO-GT Humanized Models

Metabolic Target Humanized Mouse Models

Cytokine Gene Humanized Mouse Models

Fat Reduction and Muscle Gain

Atherosclerosis

Small Nucleic Acids

구매 가능한 제품 종류

연령

Genotype

성별

수량

가격 문의

HUGO-GT Humanized Models

Metabolic Target Humanized Mouse Models

Cytokine Gene Humanized Mouse Models

Fat Reduction and Muscle Gain

Atherosclerosis

Small Nucleic Acids

기본 정보

검증 데이터

huPCSK9 mice is 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. In addition, Cyagen has developed a similar model, the B6-hPCSK9(CDS) mouse (PCSK9 coding sequence humanized model, Catalog Number: C001593). Compared to the huPCSK9 mouse model, the B6-hPCSK9(CDS) mouse expresses higher levels of human PCSK9 and exhibits LDLR protein expression closer to physiological levels. It is recommended to choose the appropriate model based on the type of drug or research direction.

Reference

Melendez QM, Krishnaji ST, Wooten CJ, Lopez D. Hypercholesterolemia: The role of PCSK9. Arch Biochem Biophys. 2017 Jul 1;625-626:39-53.

Seidah NG, Awan Z, Chrétien M, Mbikay M. PCSK9: a key modulator of cardiovascular health. Circ Res. 2014 Mar 14;114(6):1022-36.

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.

Sabatine MS. PCSK9 inhibitors: clinical evidence and implementation. Nat Rev Cardiol. 2019 Mar;16(3):155-165.

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.

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.

Kasichayanula S, Grover A, Emery MG, Gibbs MA, Somaratne R, Wasserman SM, Gibbs JP. Clinical Pharmacokinetics and Pharmacodynamics of Evolocumab, a PCSK9 Inhibitor. Clin Pharmacokinet. 2018 Jul;57(7):769-779.

Lamb YN. Inclisiran: First Approval. Drugs. 2021 Feb;81(3):389-395.

변형 전략

The mouse Pcsk9 gene sequence was replaced with the corresponding sequences in the human PCSK9 gene, including the UTR regions.

Figure 1. Gene editing strategy of huPCSK9 mice.

응용 분야

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.

검증 데이터

1. Expression of human PCSK9 gene and mouse Pcsk9 gene

RT-qPCR analysis revealed significant expression of the human PCSK9 gene in the liver and colon of homozygous huPCSK9 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 huPCSK9 mice. Notably, female huPCSK9 mice exhibited higher expression levels of the human PCSK9 gene in both the liver and colon than their male counterparts. (ND: Not detected)

Figure 2. Expression of human PCSK9 and mouse Pcsk9 genes in the liver and colon of 6-week-old homozygous huPCSK9 and wild-type (WT) mice (n=4).

2. Expression of human PCSK9 protein

（1）Western Blots

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 huPCSK9 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 huPCSK9 mice successfully express both forms of human PCSK9 protein.

Figure 3. Expression of human PCSK9 protein (hPCSK9) in the liver and colon tissues of wild-type (WT) and huPCSK9 mice. *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.

（2）ELISA

Human-specific antibodies were utilized in ELISA assays to detect the presence of human PCSK9 protein in the serum of WT and huPCSK9 mice. The results revealed significant expression of human PCSK9 protein in the serum of huPCSK9 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)

Figure 4. Expression of human PCSK9 protein in wild-type (WT) and huPCSK9 mice serum.

3. Stable expression of human PCSK9 protein

The results showed that huPCSK9 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)

Figure 5. ELISA of human PCSK9 protein in serum of huPCSK9 mice at different age stages (n=5).

4. Blood Biochemistry

The results show that the levels of T-CHO, HDL-C, LDL-C, and AST in huPCSK9 mice are significantly lower than those in WT mice, while the levels of TG and ALT show no significant differences. (Mice were not fasted before blood collection; *p<0.05; **p<0.01; ***p<0.001; Bars represent mean ± SEM)

Figure 6. Blood biochemical test results of 11-week-old homozygous huPCSK9 mice and wild-type (WT) mice (n=5). * TG: Triglycerides; T-CHO: Total cholesterol; HDL-C: High-density lipoprotein cholesterol; LDL-C: Low-density lipoprotein cholesterol; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase.

5. Expression of the mouse Ldlr gene

RT-qPCR results show that the expression level of the mouse Ldlr gene in the liver of huPCSK9 mice is higher than that in wild-type mice.

Figure 7. Expression of the mouse Ldlr gene in the liver of 6-week-old homozygous huPCSK9 and wild-type (WT) mice (n=4).

6. Expression of the mouse LDLR protein

The results from Western Blotting indicated that LDLR protein expression levels in the livers of huPCSK9 mice were slightly elevated compared to wild-type mice. No significant difference in hepatic LDLR protein expression was observed between female and male huPCSK9 mice.

Figure 8. Western Blot analysis of LDLR protein expression in the livers of 6-week-old homozygous huPCSK9 mice and wild-type (WT) mice.

7. Drug Efficacy Validation

（1）High-Fat, High-Cholesterol Diet Feeding

Mice were acclimated to a high-fat, high-cholesterol diet for 1 week starting at 5 weeks of age, followed by a switch to a formal WD diet. WD feeding resulted in a significant increase in body weight in both huPCSK9 and WT mice (Bars represent mean ± SD, n≥8).

Figure 9. Body weight changes in huPCSK9 and wild-type (WT) male mice following a high-fat, high-cholesterol diet (WD).

（2）Evolocumab Efficacy Validation

a. Efficacy validation study design: Mice were acclimated to a high-fat, high-cholesterol diet (WD) for 1 week starting at 5 weeks of age. From 6 weeks of age, mice were placed on a formal high-fat, high-cholesterol diet for 12 weeks. After 12 weeks on the formal WD diet, a single dose of Evolocumab (20mg/kg) was administered via tail vein injection for therapeutic intervention. To assess efficacy, blood samples were collected via retro-orbital bleeding under non-fasting conditions at 9 days prior to, and 2, 5, 8, 12, and 19 days post-Evolocumab administration. Serum was then harvested for subsequent analysis.

b. Serum LDL-C levels: Following Evolocumab administration, serum LDL-C levels in huPCSK9+WD mice were significantly reduced compared to WT+WD mice, while no significant alteration in LDL-C levels was observed in WT+WD mice (Bars represent mean ± SEM, n≥4).

c. Serum PCSK9 levels: At 5 days post-Evolocumab injection, serum human PCSK9 levels in huPCSK9+WD mice did not exhibit a significant decrease. This observation aligns with the anticipated mechanism of action for Evolocumab (Bars represent mean ± SEM, n=3).

Drug Information: Evolocumab functions by specifically binding to PCSK9 protein, thereby sterically hindering its interaction with LDLR. This blockade increases the quantity of LDLR receptors on the hepatocyte surface available for clearing LDL-C from the circulation, ultimately leading to a reduction in LDL-C levels. Consequently, Evolocumab has a relatively minor impact on overall PCSK9 protein levels ^[7].

Figure 10. Evolocumab efficacy validation study design and results.

（3）Inclisiran Efficacy Validation

a. Efficacy validation study design: Mice were acclimated to a high-fat, high-cholesterol diet (WD) for 1 week commencing at 5 weeks of age. From 6 weeks of age, mice were maintained on a formal high-fat, high-cholesterol diet for 12 weeks. Following 12 weeks of formal WD feeding, a single dose of Inclisiran (3mg/kg) was administered via subcutaneous injection to initiate therapeutic intervention. To evaluate drug efficacy, blood samples were collected via retro-orbital bleeding under non-fasting conditions at 9 days prior to, and 5, 12, 19, and 26 days post-Inclisiran administration. Serum was then harvested for downstream analyses. Furthermore, at 19 days post-injection, livers from a subset of mice were harvested for Western Blot and RT-qPCR analyses.

b. Serum LDL-C levels: The data revealed that following Inclisiran administration, serum LDL-C levels in huPCSK9+WD mice exhibited a reduction compared to WT+WD mice, whereas no marked change in LDL-C levels was observed in WT+WD mice (Bars represent mean ± SEM, n≥4).

c. Serum PCSK9 levels: At 19 days following Inclisiran injection, serum human PCSK9 levels in huPCSK9+WD-treated mice were significantly decreased, indicating that Inclisiran effectively suppressed the synthesis of human PCSK9 protein (Bars represent mean ± SEM, n≥4).

d. Liver PCSK9 levels: Liver PCSK9 levels in huPCSK9+WD-treated mice were markedly reduced at 19 days post-Inclisiran injection, confirming that Inclisiran effectively inhibited hepatic PCSK9 protein synthesis (the band at approximately 70KD represents a non-specific band).

Drug Information: Inclisiran is a long-acting RNA interference therapeutic that functions by targeting human PCSK9 mRNA, thereby diminishing hepatic PCSK9 protein synthesis. This reduction in PCSK9 protein levels in vivo ultimately enhances the liver's capacity to clear LDL-C cholesterol ^[8].

Figure 11. Inclisiran efficacy validation study design and results.

（4）Small Interfering RNA (siRNA) Efficacy Validation

Four siRNA drugs were administered via single subcutaneous injection at a dose of 5 mpk. Enzyme-linked immunosorbent assay (ELISA) was used to monitor changes in serum hPCSK9 protein levels in huPCSK9 mice after treatment. ELISA results showed that all four drugs inhibited hPCSK9 protein expression to varying degrees. The left panel illustrates changes in serum hPCSK9 protein levels post-treatment, while the right panel displays the ratio of hPCSK9 protein levels relative to day 0 (D0) on different days after treatment (with each mouse’s D0 hPCSK9 level set as 1). (Bars represent mean ± SEM, n=3)

Figure 12. Changes in serum human PCSK9 protein levels in huPCSK9 mice treated with siRNA drugs (6-week-old, male, serum)*. *Data provided by the client. The four siRNA drugs (PC1, PC2, PC3, PC4), supplied by the client, are designed to inhibit hPCSK9 expression.

huPCSK9 Mouse

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Reference

변형 전략

응용 분야

1. Expression of human PCSK9 gene and mouse Pcsk9 gene

2. Expression of human PCSK9 protein

3. Stable expression of human PCSK9 protein

4. Blood Biochemistry

5. Expression of the mouse Ldlr gene

6. Expression of the mouse LDLR protein

7. Drug Efficacy Validation

관련 제품