Slc26a4 in to the PL253 plasmid (Fig. 1A). The subcloned genomic 12.8-kb area was modified in a subsequent targeting round by inserting the neomycin (neo) cassette from the PL452 plasmid and developing the c.2168A.G mutation in exon 19. The targeting vector was then linearized by NotI digestion and electroporated into R1 embryonic stem (ES) cells. G418 (240 mg/mL) and ganciclovir (2 mM) double-resistant clones were analyzed by Southern blot hybridization (Fig. 1B).Figure 1. Generation of mice using the Slc26a4 p.H723R mutation. (A) Targeting scheme. A BAC clone (clone no. bMQ-323G13 GeneserviceTM) from the 129S7/AB2.2 BAC library containing the mouse Slc26a4 genomic area was used to construct the targeting vector. (1) Restriction map from the wild-type genomic mouse Slc26a4 locus.(S)-2-(3-Bromophenyl)pyrrolidine Order The expected size of your XbaI restriction fragment was 12.eight kb. (two) Targeting vector (Television) construction. The loxP-flanked neomycin resistance gene (neo) was made use of as a choice marker through embryonic stem (ES) cell culture. The c.2168 A.G mutation in exon 19 is labeled with a star. (3?) The targeted locus was introduced, and after that, the neo cassette was removed. LA, long arm; SA, brief arm. (B) Southern blot evaluation of ES cell clones. Genomic DNA from two targeted and two wild-type clones had been digested with XbaI and hybridized using the probe to verify the targeting event. (C) DNA sequencing of Slc26a4+/+ and Slc26a4 tm2Dontuh/tm2Dontuh mice. The electrophoretogram shows the p.H723R mutation. The A to G mutation (arrow) at position 2168 led to the replacement of a histidine (His, H) residue at position 723 with arginine (Arg, R). doi:10.1371/journal.pone.0064906.gPLOS 1 | plosone.orgMouse Model with SLC26A4 p.H723R MutationThe retained neo cassette flanked by loxP internet sites was excised in vivo by transfecting the targeted clone with plasmid transiently expressing the Cre recombinase. Established ES clones were then identified by polymerase chain reaction (PCR) screening and subsequently injected into C57BL/6 blastocysts to generate chimeras. After germline transmission with the targeted mutation allele, we developed the congenic Slc26a4+/tm2Dontuh mouse line applied within this study by repeated backcrossing into the C57BL/6 inbred strain for 6?0 generations, immediately after which mice homozygous for the mutation (i.Pent-2-ynoic acid web e.PMID:33719919 , Slc26a4tm2Dontuh/tm2Dontuh) had been obtained by intercrossing heterozygous mice (i.e., Slc26a4+/tm2Dontuh) (Fig. 1C). Reverse transcriptionPCR (RT-PCR) of mRNA of inner ear extract followed by direct sequencing also indicated a pure non-chimeric genetic background without unintentionally wild-type Slc26a4 expression in Slc26a4tm2Dontuh/tm2Dontuh mice. Corresponding towards the human genotypes, mice with compound heterozygous mutations for p.H723R and c.9192A.G (i.e., Slc26a4tm1Dontuh/tm2Dontuh) had been also generated by intercrossing heterozygous Slc26a4+/tm2Dontuh mice with Slc26a4tm1Dontuh/tm1Dntuh mice. All animal experiments have been carried out in accordance with animal welfare suggestions and approved by the Institutional Animal Care and Use Committee (IACUC) of National Taiwan University College of Medicine (approval no. 20110123).Audiological and Vestibular EvaluationsFor audiological evaluations, the mice had been anesthetized with sodium pentobarbital (35 mg/kg) delivered intraperitoneally and placed in a head-holder within an acoustically and electrically insulated and grounded test room. We applied an evoked potential detection program (Intelligent EP 3.90; Intelligent Hearing Systems, Miami,.
