65 TWO NOVEL, CRISPR-DERIVED FGF10 KNOCKOUT MOUSE STRAINS: A MODEL FOR DUODENAL ATRESIA RESEARCH
Warwick Teague1,2,3, Angelique Catubig1, Sebastian King1,2,3, Ruili Li1, John Hutson1,2,3
1Murdoch Children's Research Institute, Melbourne, Australia. 2The Royal Children's Hospital, Melbourne, Australia. 3University of Melbourne, Melbourne, Australia

Abstract

AIM OF THE STUDY
Duodenal atresia (DA) is a congenital bowel obstruction affecting 1:7000 livebirths. The cause of DA is unknown, and Tandler’s 1900 recanalisation theory is contradicted by modern science. The genes for Fibroblast Growth Factor 10 (FGF10) and its receptor are of interest as 30-50% mice not expressing these genes also have DA. This study aimed to determine the suitability of novel mouse strains as a model for DA.

METHODS
Deletions of FGF10 gene were performed using CRISPR to generate 2 novel mouse strains. Gene sequencing and PCR methods were used to determine DNA sequence, gene and RNA expression. FGF10 heterozygote (+/-) plug mating strategy was used to generate E15.5-18.5 embryos for analysis of genotype and phenotype. This study has animal ethics (A792) and biosafety (215-2014-PC1-NLRD) approval.

MAIN RESULTS
Two novel mouse strains were derived targeting deletions on exon 3 of the FGF10 gene, here termed Strain-1 (130 base-pair deletion) and Strain-2 (13 base-pair deletion). RNA expression quantification correlated with genotype. General phenotype of FGF10 nulls was consistent with previous reports, e.g. abnormal facies, absent limbs, abnormal trachea, absent lungs and small stomach. Heterozygotes showed normal duodenal morphology. 22/106 (21%) Strain-1 embryos were nulls (-/-), including 17/22 (77%) with DA (47% type 1, 18% type 2, 35% type 3). 20/87 (23%) Strain-2 embryos were nulls, including 14/20 (70%) with DA (50% type 1, 14% type 2, 36% type 3).

CONCLUSIONS
These novel mouse strains demonstrate the genetic and phenotypic characteristics of an FGF10 knockout. Their duodenal phenotype is notable: 1) DA penetrance is increased compared with other strains, 2) each strain gives rise to all 3 DA types, 3) association between DA and tracheal anomalies is akin to human DA. Therefore, these strains present a promising animal model to further investigate DA.


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