B6-hCFTR*F508del Mice

Catalog Number: I001226

Strain Name: C57BL/6NCya-Cftrtm2(hCFTR c.1521_1523 del CTT)/Cya

Genetic Background: C57BL/6NCya

One of Cyagen's HUGO-GT™ (Humanized Genomic Ortholog for Gene Therapy) Mouse Strains

 

Strain Description

The cystic fibrosis transmembrane conductance regulator (CFTR) is a critical protein that maintains the salt and water balance across various human organs, including the lungs, pancreas, and sweat glands. The primary function of CFTR is to act as a chloride channel, regulating the transport of chloride and bicarbonate ions across epithelial cell membranes, thereby maintaining tissue fluid balance and pH. This process is ATP-dependent and also modulates the activity of other ion channels and transport proteins [1-2]. Mutations in the CFTR gene can lead to chloride channel dysfunction, resulting in various diseases, with cystic fibrosis (CF) being the most common. CF is the most prevalent lethal genetic disease among Caucasians, with an incidence of approximately 1/2,500 to 1/1,800, and about 90,000 cases globally [3-4]. The disease is characterized by thickened mucus in the lungs, frequent respiratory infections, pancreatic insufficiency, and male infertility, typically due to vas deferens obstruction. The F508del (ΔF508) mutation is the most common pathogenic mutation in CF, with about 80% of CF patients carrying at least one allele of this mutation, and approximately 40% being homozygous [5]. This mutation causes the deletion of phenylalanine (F508) in the first nucleotide-binding domain (NBD1) of the CFTR protein, leading to misfolding and endoplasmic reticulum (ER)-mediated degradation, preventing CFTR from reaching the cell membrane and compromising chloride channel function, which results in chronic pulmonary symptoms [6-7]. Current treatments for CF mainly focus on CFTR modulators to restore the function of the mutated CFTR protein. CFTR modulators are classified into potentiators (which enhance CFTR function) and correctors (which assist in the proper folding and trafficking of CFTR to the cell membrane). Representative drugs include Ivacaftor, Lumacaftor, and triple-combination CFTR modulating therapy Elexacaftor-Tezacaftor-Ivacaftor [8].

This strain was developed by introducing the F508del mutation into the CFTR-humanized mouse model (Catalog Number: I001132), creating a humanized disease model. The introduction of the mutation results in the manifestation of CF-related phenotypes in mice, making it suitable for research into CF mechanisms and the screening, development, and evaluation of therapies targeting the CFTR F508del mutation. In addition, based on the independently developed TurboKnockout fusion BAC recombination technology, Cyagen can also generate hot mutation models based on the CFTR-humanized strain and provide customized services for specific mutations to meet the experimental needs in pharmacology and other fields.

Strain Strategy

  • Construction strategy for the B6-hCFTR wild-type humanized model (Catalog Number: I001132): The region from ~9.5 kb upstream to ~1.2 kb downstream of mouse Cftr gene was replaced with the region from ~40.1 kb upstream to ~25 kb downstream of human CFTR gene.



  • Construction strategy for the B6-hCFTR*F508del point mutation humanized model (Catalog Number: I001226): Gene editing technology was used to introduce the F508del mutation (c.1521_1523 delCTT) into exon 11 of the human CFTR gene in the B6-hCFTR mouse.


Application

This strain is suitable for studying the mechanisms underlying cystic fibrosis and evaluating targeted therapies in terms of their efficacy and pharmacodynamics.

Validation Data

1. Gene Expression Detection

Figure 1. Comparison of human CFTR and mouse Cftr gene expression in tissues of wild-type (WT) mice, B6-hCFTR mice (hCFTRKI/KI), and B6-hCFTR*F508del mice (hCFTRΔF508/ΔF508). RT-qPCR results showed that human CFTR gene expression was detected in the liver, intestines, and lungs of B6-hCFTR and B6-hCFTR*F508del mice, with no endogenous mouse Cftr gene expression. The expression level of human CFTR in B6-hCFTR*F508del mice was slightly lower than in B6-hCFTR mice. (ND: Not detected)

2. Protein Expression Detection


Figure 2. Human CFTR protein expression in the colon and lung tissues of wild-type (WT) mice, B6-hCFTR mice (hCFTRKI/KI), and B6-hCFTR*F508del mice (hCFTRΔF508/ΔF508). Western Blot results showed successful expression of human CFTR protein in B6-hCFTR and B6-hCFTR*F508del mice, with the expression level in B6-hCFTR*F508del mice significantly lower than in B6-hCFTR mice.

3. Intestinal Tissue Pathology


Figure 3. Intestinal tissue pathology comparison of wild-type (WT) mice, B6-hCFTR mice (hCFTRKI/KI), and B6-hCFTR*F508del mice (hCFTRΔF508/ΔF508) at 5 weeks of age. H&E staining results showed significant pathological features in B6-hCFTR*F508del mice, including goblet cell hyperplasia (red arrows), mucus accumulation (green arrows), and increased intestinal wall thickness (black arrows). B6-hCFTR mice showed mild abnormal phenotypes, while wild-type mice had normal intestinal tissue.

4. Intestinal Wall Thickness and Goblet Cell Count


Figure 4. Comparison of intestinal wall thickness and goblet cell count in wild-type (WT) mice, B6-hCFTR mice (hCFTRKI/KI), and B6-hCFTR*F508del mice (hCFTRΔF508/ΔF508) at 5 weeks of age. Quantitative analysis showed significantly increased ileal and jejunal wall thickness and goblet cell count in B6-hCFTR*F508del mice compared to wild-type and B6-hCFTR mice.

Expanded Information: The Rare Disease Data Center (RDDC)

1. Basic information about the CFTR gene


2. CFTR clinical variants


3. Disease introduction

Cystic Fibrosis (CF) is an autosomal recessive genetic disorder that causes severe damage to the lungs, digestive system, and other organs. It thickens normal mucus, sweat, and digestive fluids  by affecting cells, preventing them from acting as lubricants and blocking ducts and channels. The impact on the lungs and pancreas is particularly severe. The disease has five clinical characteristics. Firstly, there is persistent productive cough and hyperinflation of the lung lobes in the respiratory system. Secondly, there is chronic nasal congestion, headaches, chronic postnasal drip cough, and sleep disorders in the sinuses. Thirdly, there are digestive system disorders such as pancreatic exocrine dysfunction and increased intestinal viscosity leading to intestinal obstruction. Fourthly, there are reproductive system disorders such as sperm transport defects leading to infertility in male CF patients, although spermatogenesis is unaffected. Fifthly, due to the reduced mineral content in the bones of CF patients, there may be nutritional and growth development disorders, such as an increased incidence of fractures and scoliosis.

 

4. CFTR gene and mutations

The CFTR gene encodes the Cystic Fibrosis Transmembrane Conductance Regulator, a cAMP-dependent chloride ion channel protein distributed in the airways, pancreatic ducts, digestive tract, reproductive system, sweat glands, etc., driving the secretion of chloride ions and bicarbonate. Abnormal CFTR function can cause transmembrane transport disorders of chloride ions and bicarbonate, leading to widespread mucus obstruction in exocrine glands throughout the body, affecting multiple systems such as respiration, digestion, endocrine, and reproduction. More than 2000 mutations of the CFTR gene have been discovered so far, with varying incidence rates reported in different countries and regions. The incidence rate in newborns is approximately 1/25000 to 1/1800. The number of cases in Asia and Africa is far less than in Europe and North America. In addition, 85% to 90% of Caucasian CF patients carry at least one F508del mutation [9].

 

5. Function of non-coding DNA sequences

Researchers have found numerous miRNA binding sites in the CFTR mRNA. Among them, miR-145-5p can inhibit the synthesis of CFTR through interactions with CFTR and other factors. To further investigate, researchers synthesized a peptide nucleic acid (PNAs) R8-PNA-a145 targeting miR-145-5p. Experimental results showed that this PNA can inhibit the expression of miR-145-5p and upregulate the expression of CFTR mRNA. This finding suggests that the 3’UTR of CFTR has a potential drug target value [10].

 

6. CFTR-targeted gene therapy

The current CF treatment pipeline is primarily focused on small-molecule drugs. In recent years, numerous pipelines related to gene therapy have also emerged. One such pipeline is Eluforsen, a clinical phase 1 ASO-related pipeline from ProQR company. This pipeline targets the region near the F508dcl mutation of the CFTR gene to restore the function of the CFTR protein [11-12]. The CFTR-F508del mouse model was used in its preclinical research.

In summary, the CFTR gene is a significant pathogenic gene for Cystic Fibrosis (CF). Humanizing mouse genes can help accelerate CFTR-targeted therapies into clinical stages. CFTR gene humanized mice from Cyagen can be used for preclinical research on CF and customized services can also be provided for different point mutations.

 

References
[1]Corradi V, Vergani P, Tieleman DP. Cystic Fibrosis Transmembrane Conductance Regulator (CFTR): CLOSED AND OPEN STATE CHANNEL MODELS. J Biol Chem. 2015 Sep 18;290(38):22891-906.
[2]Csanády L, Vergani P, Gadsby DC. STRUCTURE, GATING, AND REGULATION OF THE CFTR ANION CHANNEL. Physiol Rev. 2019 Jan 1;99(1):707-738. 
[3]Chillón M, Casals T, Mercier B, Bassas L, Lissens W, Silber S, Romey MC, Ruiz-Romero J, Verlingue C, Claustres M, et al. Mutations in the cystic fibrosis gene in patients with congenital absence of the vas deferens. N Engl J Med. 1995 Jun 1;332(22):1475-80.
[4]Grasemann H, Ratjen F. Cystic Fibrosis. N Engl J Med. 2023 Nov 2;389(18):1693-1707.
[5]Lopes-Pacheco M. CFTR Modulators: The Changing Face of Cystic Fibrosis in the Era of Precision Medicine. Front Pharmacol. 2020 Feb 21;10:1662.
[6]Hoelen H, Kleizen B, Schmidt A, Richardson J, Charitou P, Thomas PJ, Braakman I. The primary folding defect and rescue of ΔF508 CFTR emerge during translation of the mutant domain. PLoS One. 2010 Nov 30;5(11):e15458.
[7]He L, Skirkanich J, Moronetti L, Lewis R, Lamitina T. The cystic-fibrosis-associated ΔF508 mutation confers post-transcriptional destabilization on the C. elegans ABC transporter PGP-3. Dis Model Mech. 2012 Nov;5(6):930-9. 
[8]Valladares KN, Jones LI, Barnes JW, Krick S. Highly Effective Modulator Therapy: Implications for the Microbial Landscape in Cystic Fibrosis. Int J Mol Sci. 2024 Nov 5;25(22):11865.
[9]Zeiher B G, Eichwald E, Smith J J, et al.A MouseModelfortheAF508Allele of Cystic Fibrosis[J].[2023-07-17].
[10]Enrica F, Anna T, Tiziana J, et al. A Peptide Nucleic Acid against MicroRNA miR-145-5p Enhances the Expression of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in Calu-3 Cells[J]. Molecules, 2018, 23(1).
[11]Sermet-Gaudelus, IsabelleClancy, John P.Nichols, David P.Nick, Jerry A.De Boeck, KrisSolomon, George M.Mall, Marcus A.Bolognese, JamesBouisset, Florileneden Hollander, WilhelminaPaquette-Lamontagne, NicolasTomkinson, NigelHenig, NoreenElborn, J. StuartRowe, Steven M.Antisense oligonucleotide eluforsen improves CFTR function in F508del cystic fibrosis[J]. Journal of cystic fibrosis: official journal of the European Cystic Fibrosis Society, 2019, 18(4).
[12]Beumer W, Swildens J, Leal T, et al. Evaluation of eluforsen, a novel RNA oligonucleotide for restoration of CFTR function in in vitro and murine models of p.Phe508del cystic fibrosis[J].PLoS ONE, 2019, 14(6):e0219182-.