FVB-Abcb4 KO Mice

Catalog Number: C001590

Strain Name: FVB/NJCya-Abcb4em1/Cya

Genetic Background: FVB/NJCya

Reproduction: Homozygote x Homozygote

 

Strain Description

Progressive Familial Intrahepatic Cholestasis Type 3 (PFIC3) is a rare, life-threatening autosomal recessive hereditary liver disease caused by mutations in the ABCB4 gene (also known as MDR3 in humans and MDR2 in rodents) [1-3]. The disease is characterized by early persistent cholestasis, leading to the accumulation of bile acids in the liver and subsequent hepatocellular damage. Clinical manifestations of PFIC3 include jaundice (yellowing of the skin and eyes), pruritus, fatigue, and growth failure. If untreated, it can progress to cirrhosis and liver failure in early childhood [4]. The ABCB4 gene encodes Multidrug Resistance Protein 3 (MDR3), a member of the ATP-binding cassette (ABC) transporter family, which is a liver-specific phosphatidylcholine (PC) transporter [5]. MDR3 is primarily expressed on the canalicular membrane of hepatocytes (the membrane that forms bile canaliculi). It is involved in the transport of phosphatidylcholine from the hepatocyte membrane to vesicles, which are then secreted into bile, forming PC-cholesterol vesicles and a small number of mixed bile salt micelles [6]. Mutations in ABCB4 lead to the loss or dysfunction of MDR3, reducing PC levels in bile, destabilizing micelles, and increasing bile salt concentrations, thereby causing cholestasis and hepatocellular injury [7].

Studies have shown that knocking out the Abcb4 gene in mice leads to a phenotype similar to human PFIC3, although the severity and progression of the disease vary across different mouse strains. In the commonly used C57BL/6 background, Abcb4-KO mice exhibit a relatively mild pathological phenotype due to lower bile salt toxicity, and a diet enriched in hydrophobic bile salts is typically required to induce a more human-like PFIC3 phenotype [8-10]. In contrast, Abcb4-KO mice in the FVB background naturally exhibit most of the biomarkers and pathological features of human PFIC3, including hepatomegaly, liver fibrosis, and early disease onset with more severe progression [10-11].

Cyagen has generated the FVB-Abcb4 KO mouse model by knocking out the Abcb4 gene in FVB mice. This model lacks the Abcb4 gene and protein expression and exhibits liver enlargement, elevated liver function markers, and increased total bilirubin. Histopathological examination shows hepatocyte necrosis, inflammatory cell infiltration, connective tissue proliferation, bile duct proliferation, and liver fibrosis.

Strain Strategy


Figure 1. Schematic of the gene editing strategy for FVB-Abcb4 KO mice. The Abcb4 gene in FVB mice is knocked out by targeting exons 9 to 12 using gene editing techniques.

 

Strain Application

  • Study of Multidrug Resistance Protein 3 (MDR3) function;
  • Mechanistic studies of Progressive Familial Intrahepatic Cholestasis Type 3 (PFIC3);
  • Drug development, screening, and preclinical efficacy evaluation for PFIC3 treatment.

 

Validation Data

1. Gene Expression Detection


Figure 2. Abcb4 gene expression detection in various tissues of FVB wild-type (WT) and FVB-Abcb4 KO mice (6 weeks of age). RT-qPCR results show that Abcb4 gene expression is undetectable in the heart, spleen, and liver of FVB-Abcb4 KO mice, confirming successful gene knockout (Bars represent mean ± SEM, n=3).

 

2. Protein Expression Detection

Figure 3. ABCB4 protein expression detection in the liver of FVB wild-type (WT) and FVB-Abcb4 KO mice (6 weeks of age). Western Blot analysis shows no ABCB4 protein expression in the liver of FVB-Abcb4 KO mice.

 

3. Liver Morphology


Figure 4. Comparison of liver morphology between FVB wild-type (WT) and FVB-Abcb4 KO mice (6 weeks of age). The liver of FVB-Abcb4 KO mice is significantly enlarged compared to wild-type mice.

 

4. Liver Function Tests


Figure 5. Comparison of liver function markers between FVB wild-type (WT) and FVB-Abcb4 KO mice (6 weeks of age). FVB-Abcb4 KO mice show significantly elevated alanine aminotransferase (ALT) and Aspartate transaminase (AST), indicating hepatocellular damage. Alkaline phosphatase (ALP) and total bilirubin (T-BIL) levels are also significantly elevated, suggesting cholestatic jaundice (Bars represent mean ± SEM, n=5).

 

5. H&E Staining


Figure 6. Comparison of liver H&E staining and pathological scores between FVB wild-type (WT) and FVB-Abcb4 KO mice. In contrast to wild-type mice, the liver of FVB-Abcb4 KO mice shows disrupted hepatocyte structure around the portal areas, hepatocyte necrosis (black arrow), connective tissue proliferation (green arrow), fibrous septa formation, and inflammatory cell infiltration (red arrow), along with notable bile duct proliferation (blue arrow). Pathological scores show significantly higher bile duct proliferation and focal inflammatory cell infiltration in FVB-Abcb4 KO mice compared to wild-type mice.

ND: Not detected.

 

6. Masson Staining


Figure 7. Comparison of liver Masson staining between FVB wild-type (WT) and FVB-Abcb4 KO mice. Masson staining, which highlights collagen fibers, shows no significant collagen fiber proliferation in the liver of wild-type mice. In contrast, FVB-Abcb4 KO mice exhibit pronounced fibrosis and collagen fiber proliferation (black arrow) around the portal areas, fibrous septa formation, and fibrous bridging between adjacent portal areas.

 

References
[1]Davit-Spraul A, Gonzales E, Baussan C, Jacquemin E. Progressive familial intrahepatic cholestasis. Orphanet J Rare Dis. 2009 Jan 8;4:1.
[2]Vij M, Safwan M, Shanmugam NP, Rela M. Liver pathology in severe multidrug resistant 3 protein deficiency: a series of 10 pediatric cases. Ann Diagn Pathol. 2015 Oct;19(5):277-82.
[3]de Vree JM, Jacquemin E, Sturm E, Cresteil D, Bosma PJ, Aten J, Deleuze JF, Desrochers M, Burdelski M, Bernard O, Oude Elferink RP, Hadchouel M. Mutations in the MDR3 gene cause progressive familial intrahepatic cholestasis. Proc Natl Acad Sci U S A. 1998 Jan 6;95(1):282-7.
[4]Smit JJ, Schinkel AH, Oude Elferink RP, Groen AK, Wagenaar E, van Deemter L, Mol CA, Ottenhoff R, van der Lugt NM, van Roon MA, et al. Homozygous disruption of the murine mdr2 P-glycoprotein gene leads to a complete absence of phospholipid from bile and to liver disease. Cell. 1993 Nov 5;75(3):451-62.
[5]Delaunay JL, Durand-Schneider AM, Dossier C, Falguières T, Gautherot J, Davit-Spraul A, Aït-Slimane T, Housset C, Jacquemin E, Maurice M. A functional classification of ABCB4 variations causing progressive familial intrahepatic cholestasis type 3. Hepatology. 2016 May;63(5):1620-31.
[6]Oude Elferink RP, Paulusma CC, Groen AK. Hepatocanalicular transport defects: pathophysiologic mechanisms of rare diseases. Gastroenterology. 2006 Mar;130(3):908-25.
[7]Morotti RA, Suchy FJ, Magid MS. Progressive familial intrahepatic cholestasis (PFIC) type 1, 2, and 3: a review of the liver pathology findings. Semin Liver Dis. 2011 Feb;31(1):3-10.
[8]Smit JJ, Schinkel AH, Oude Elferink RP, Groen AK, Wagenaar E, van Deemter L, Mol CA, Ottenhoff R, van der Lugt NM, van Roon MA, et al. Homozygous disruption of the murine mdr2 P-glycoprotein gene leads to a complete absence of phospholipid from bile and to liver disease. Cell. 1993 Nov 5;75(3):451-62.
[9]Ikenaga N, Liu SB, Sverdlov DY, Yoshida S, Nasser I, Ke Q, Kang PM, Popov Y. A new Mdr2(-/-) mouse model of sclerosing cholangitis with rapid fibrosis progression, early-onset portal hypertension, and liver cancer. Am J Pathol. 2015 Feb;185(2):325-34.
[10]Weber ND, Odriozola L, Martínez-García J, Ferrer V, Douar A, Bénichou B, González-Aseguinolaza G, Smerdou C. Gene therapy for progressive familial intrahepatic cholestasis type 3 in a clinically relevant mouse model. Nat Commun. 2019 Dec 13;10(1):5694.
[11]Aronson SJ, Bakker RS, Shi X, Duijst S, Ten Bloemendaal L, de Waart DR, Verheij J, Ronzitti G, Oude Elferink RP, Beuers U, Paulusma CC, Bosma PJ. Liver-directed gene therapy results in long-term correction of progressive familial intrahepatic cholestasis type 3 in mice.