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Editing Myosin VB Gene to Create Porcine Model of Microvillus Inclusion Disease, With Microvillus-Lined Inclusions and Alterations in Sodium Transporters.
- Source :
-
Gastroenterology [Gastroenterology] 2020 Jun; Vol. 158 (8), pp. 2236-2249.e9. Date of Electronic Publication: 2020 Feb 26. - Publication Year :
- 2020
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Abstract
- Background & Aims: Microvillus inclusion disease (MVID) is caused by inactivating mutations in the myosin VB gene (MYO5B). MVID is a complex disorder characterized by chronic, watery, life-threatening diarrhea that usually begins in the first hours to days of life. We developed a large animal model of MVID to better understand its pathophysiology.<br />Methods: Pigs were cloned by transfer of chromatin from swine primary fetal fibroblasts, which were edited with TALENs and single-strand oligonucleotide to introduce a P663-L663 substitution in the endogenous swine MYO5B (corresponding to the P660L mutation in human MYO5B, associated with MVID) to fertilized oocytes. We analyzed duodenal tissues from patients with MVID (with the MYO5B P660L mutation) and without (controls), and from pigs using immunohistochemistry. Enteroids were generated from pigs with MYO5B(P663L) and without the substitution (control pigs).<br />Results: Duodenal tissues from patients with MVID lacked MYO5B at the base of the apical membrane of intestinal cells; instead MYO5B was intracellular. Intestinal tissues and derived enteroids from MYO5B(P663L) piglets had reduced apical levels and diffuse subapical levels of sodium hydrogen exchanger 3 and SGLT1, which regulate transport of sodium, glucose, and water, compared with tissues from control piglets. However, intestinal tissues and derived enteroids from MYO5B(P663L) piglets maintained CFTR on apical membranes, like tissues from control pigs. Liver tissues from MYO5B(P663L) piglets had alterations in bile salt export pump, a transporter that facilitates bile flow, which is normally expressed in the bile canaliculi in the liver.<br />Conclusions: We developed a large animal model of MVID that has many features of the human disease. Studies of this model could provide information about the functions of MYO5B and MVID pathogenesis, and might lead to new treatments.<br /> (Copyright © 2020 AGA Institute. Published by Elsevier Inc. All rights reserved.)
- Subjects :
- Animals
Animals, Genetically Modified
Cells, Cultured
Coculture Techniques
Cystic Fibrosis Transmembrane Conductance Regulator genetics
Cystic Fibrosis Transmembrane Conductance Regulator metabolism
Disease Models, Animal
Duodenum pathology
Genetic Predisposition to Disease
Humans
Intestinal Mucosa pathology
Malabsorption Syndromes metabolism
Malabsorption Syndromes pathology
Microvilli genetics
Microvilli metabolism
Mucolipidoses metabolism
Mucolipidoses pathology
Mutation, Missense
Phenotype
Sodium metabolism
Sodium-Glucose Transporter 1 genetics
Sodium-Hydrogen Exchanger 3 genetics
Sus scrofa
Duodenum metabolism
Gene Editing
Intestinal Mucosa metabolism
Malabsorption Syndromes genetics
Microvilli pathology
Mucolipidoses genetics
Myosin Heavy Chains genetics
Myosin Type V genetics
Sodium-Glucose Transporter 1 metabolism
Sodium-Hydrogen Exchanger 3 metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1528-0012
- Volume :
- 158
- Issue :
- 8
- Database :
- MEDLINE
- Journal :
- Gastroenterology
- Publication Type :
- Academic Journal
- Accession number :
- 32112796
- Full Text :
- https://doi.org/10.1053/j.gastro.2020.02.034