Production of CFTR-ΔF508 Rabbits

Abstract

Cystic Fibrosis (CF) is a lethal autosomal recessive disease caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). The most common mutation is the deletion of phenylalanine residue at position 508 (ΔF508). Here we report the production of CFTR-ΔF508 rabbits by CRISPR/Cas9-mediated gene editing. After microinjection and embryo transfer, 77 kits were born, of which five carried the ΔF508 mutation. To confirm the germline transmission, one male ΔF508 founder was bred with two wild-type females and produced 16 F1 generation kits, of which six are heterozygous ΔF508/WT animals. Our work adds CFTR-ΔF508 rabbits to the toolbox of CF animal models for biomedical research.

Keywords: CFTR-ΔF508; CRISPR/Cas9; cystic fbrosis; gene edit; rabbits.

FIGURE 1

Design and validation of guide RNAs. (A) illustration of gRNA design. Blue box: sgRNA sequence. Bold letters within the blue box: PAM sequence. Yellow box: the F508 or the ΔF508 sequence. (B) In vitro validation results of sg01 and sg02.

FIGURE 2

Production of ΔF508 founder rabbits. (A) Summary of embryo transfer and genotyping results. (B) One founder ΔF508 kit. (C) Summary of breeding outcome to generate F1 generation ΔF508 rabbits. #Indel only refer to number of animals that carry non-ΔF508 indel mutations. #ΔF508 refers to number of animals that carry ΔF508 allele.

FIGURE 3

Histopathology of the tracheal and lungs of a heterozygous compound ΔF508/KO rabbit. (A) Illustration of allele sequence of the ΔF508/KO rabbit. Red letters indicate positions of mutations. (B) The submucosa of the trachea was thickened with moderate to marked amounts of edema (arrowheads) and submucosal blood vessels were markedly congested (asterisk). (C) tracheal epithelium was markedly flattened and attenuated (arrowhead). (D) Lung contained variable amounts of fibrin within alveoli (arrowhead), variable amounts of edema, and multifocal rod-shaped bacterial colonies [(E), arrowhead].