Generation and Characterization of RecQL4 Mouse Models

Recql4^K525A and Recql4^G522Efs mice were generated using Cripsr/Cas9 methods by the Mouse Engineering at Garvan/ABR (MEGA) services (Garvan Institute, Darlinghurst, Australia). Lysine 525 was mutated to Alanine (AAG>GCA) in single cell C57Bl/6 embryos via sgRNA-directed gene targeting and homologous recombination with a single stranded DNA oligonucleotide substrate. Viable pups were screened by DNA sequencing and one C57Bl/6 male carrying the K525A mutation on one allele and a 2bp insertion (GA) after the T521 codon (G522Efs) on the other allele was identified as a founder. The chemically (ENU) induced Recql4^M789K and Recql4^R347X mutations were obtained from the Australian Phenomics Facility (APF, Canberra, Australia: IGL01381 and IGL01809). Recql4^fl/fl mice (C57BL/6-Recql4^tm2272Arte) have been previously described [20 (link), 44 (link)]. Rosa26-CreER^T2 mice on a C57Bl/6 background were purchased from The Jackson Laboratory (B6.129-Gt(ROSA)26Sor^{tm1(cre/ERT2)Tyj}/J, Stock Number: 008463) and have been previously described [20 (link)]. All lines were on a backcrossed C57Bl/6 background. ENU mutants were outcrossed at least 6 times and assessed across multiple generations to eliminate effects of any additional mutations. Tamoxifen containing food was prepared by Specialty Feeds (Perth, Australia) at 400mg/kg Tamoxifen citrate (Sigma Aldrich) in a base of standard mouse chow.