Development of a humanised cystic fibrosis mouse model
(2019) European Cystic Fibrosis Society 16th ECFS Basic Science Conference — Location: Dubrovnik, Croatia (27.March.2019)
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Mottais, Angélique 
- Abstract
- Animal models are widely used for the study of the pathophysiology of human diseases and the efficacy of new therapies. In the context of cystic fibrosis (CF), several animal (mouse, pig or ferret) models have been developed. However, they show little or no severe respiratory phenotype typically associated with the high morbidity and mortality of the human disease. Based on the observation that the F508del mutation of CFTR leads to a more severe CF phenotype for the human CFTR (hCFTR) than the murine CFTR (mCFTR), this project aims at developing a CF mouse model expressing the hCFTR gene. A KO-CFTR mouse line was generated using the CRISPR/Cas9 genome editing system. In this aim, a guide RNA targeting the exon 1 was used to produce mutations in the mCFTR gene. In parallel, a wild-type (WT) or mutated (F508del mutation) hCFTR overexpressing mouse line was created by transgene addition. For this purpose, a plasmid construct (pcDNA3.1-hCFTR, CMV promoter) was designed, amplified, purified and microinjected into C57BL/6NCrl mouse embryos. Regarding the development of the "knock-out" mCFTR line, 33 mice were generated. After comparing the WT mouse exon 1 sequences with those of the generated mice, 7 mice were shown to have at least one mCFTR mutation. Regarding the humanized mouse model, the constructs of pcDNA3.1-hCFTR for WT and for F508del were injected into 178 and 115 embryos of C57BL/6NCrl mice respectively. Twenty-seven newborns were obtained for the WT group and 14 for the F508del group. Of these, 5 transgenic mice (2 WT-hCFTR and 3 F508del-hCFTR) were obtained. Biochemical studies using immuno-tagging and western blotting are ongoing to check whether the generated founding mice of the KO-CFTR line are indeed devoid of the expression of the CFTR protein. Quantification of the WT or the F508del mutated CFTR is to be analyzed in the founding mice of the humanized lines to check protein overexpression. When these data were verified, the founding lines will be used to generate lineages of the humanized CF mouse model, which will then be characterized from organ and tissue phenotypic point of view. The model will hopefully allow a better understanding of the genotype/phenotype differences between the hCFTR and the mCFTR.
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Citations
Mottais, A., Achouri, Y., Beka, M., Reynaerts, A., Delion, M., & Leal, T. (2019). Development of a humanised cystic fibrosis mouse model. European Cystic Fibrosis Society 16th ECFS Basic Science Conference, Dubrovnik, Croatia. https://hdl.handle.net/2078.5/28258