IDDoR: a novel reporter mouse system for simultaneous and quantitative in vivo analysis of both DNA double-strand break repair pathways

1 Shanghai Key Laboratory of Maternal Fetal Medicine, Clinical and Translational Research Center of Shanghai First Maternity and Infant Hospital, Frontier Science Center for Stem Cell Research, School of Life Sciences and Technology, Tongji University, Shanghai 200092, China 2 Tsingtao Advanced Research Institute, Tongji University, Qingdao 266071, China 3 State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China


Materials and Methods
Animals C57BL/6 mice were housed in a specific pathogen-free (SPF)-grade environment. All mice were maintained under a 12-hour light/dark cycle and had free access to food and water. All experiments were performed in accordance with the Health Guide for the Care and Use of Laboratory Animals and were approved by the Biological Research Ethics Committee of Tongji University.

Generation of dual fluorescence-based reporter mice (Fireworker mice)
The targeting vector was modified from a previously reported NHEJ-I vector. Briefly, an ATG-less tdTomato ORF was inserted to the site downstream of the second exon of GFP. Then, the first exon of GFP was replaced with the HPRT exon, and the second exon of GFP was replaced with the ATG-less GFP ORF. A single nucleotide was then added to Ad, SA was inserted into the 5' end of the HPRT exon, and the starting codon, ATG, was inserted into the 3' end of the HPRT exon. Finally, the modified repair cassette was digested and ligated to the backbone of a previously published R26NHEJ targeting vector (Vaidya et al., 2014). The primers used for genotyping were as follows: P1-5' GGGGCGTGCTGAGCCAGACCTCCAT 3'; P2-5' TCCCGACAAAACCGAAAATCTGTGG 3'; P3-5' TGCATCGCATTGTCTGAGTAGG 3'. The wild-type allele can be amplified by P1 and P2, generating a 435 bp band, and the knocked-in allele can be amplified by P2 and P3, generating a 303 bp band.

Generation of inducible I-SceI mice (Breaker mice)
The targeting vector was modified from a previously reported inducible I-SceI expression vector. Two tandem insulators were added upstream and downstream of the inducible I-SceI expression element by in-fusion cloning to avoid any potential influence of flanking sequences. The targeting vectors contained a 5 kb 5' homologous arm and a 3.8 kb 3' homologous arm targeting the mouse H11 locus. The vector was microinjected along with the in vitro transcribed Cas9 mRNA and gRNA into C57BL/6 zygotes to generate founder mice. The primers used for genotyping were as follows: P1-5' ATAAGCCATTCTCCATTTCATAA 3'; P2-5' CCCCTTGTTCCCTTTCTGC 3'; P3-5' CCGTCGAGGCTTGGGTGATA 3'; P4-5' CATGATTAGTGTTTGCCTTTGTTA 3'. The wild-type allele can be amplified by P1 and P2, generating a 428 bp band, and the knocked-in allele can be amplified by P3 and P4, generating a 278 bp band.

In vivo analysis of DNA double-strand break repair efficiency
Rosa26 Fireworker/+ mice were mated with H11 Breaker/+ mice to obtain IDDoR mice. The IDDoR mice were fed water supplemented with 3 mg/mL doxycycline and 5% sucrose for 3 weeks and then sacrificed to dissect different organs, including the heart, kidney, pancreas, stomach, intestine, brain and skin. Organs were immediately fixed with 4% paraformaldehyde for immunofluorescence or frozen at -80°C for genomic DNA and RNA extraction. For each mouse, at least six frozen sections were counted for analyzing GFP, tdTomato and Ki67 positive cells.

Mental stress induction
Mental stress was induced using a restraint model as previously reported (Tye et al., 2013;Wu et al., 2016;Zhang et al., 2020). Briefly, 6-week-old mice were individually placed inside 50 mL plastic centrifuge tubes with small punctures for two hours/day. Six-weekold control mice were maintained away from food and water but allowed free activities and contact with each other in their original cages for two hours/day. The induction period lasted for a total of 14 consecutive days.

Immunofluorescence histochemistry
The freshly dissected tissues were fixed with 4% paraformaldehyde at 4 °C overnight, washed with PBS 3 times and treated with 30% sucrose/PBS solution for cryopreservation until the tissues sank. Then, the tissues were embedded in Tissue-Tek ® O.C.T. compound and sectioned with Leica CM1950 cryostats. The sections were washed with PBS three times and incubated in 0.5% Triton-X-100/PBS solution for 10 minutes. After rinsing with PBS 3 times, the sections were blocked with 2% BSA/PBS for 1 hour at room temperature and then incubated with primary antibodies at 4 °C overnight. The next day, the sections were rinsed with PBS 3 times and incubated with secondary antibodies at room temperature for 1 hour. The sections were then covered with mounting medium containing DAPI nuclear staining followed by PBS washing 3 times. For each mouse, at least six frozen sections were counted.

Real-time PCR
For copy number determination of the Fireworker vector, genomic DNA was prepared from the mouse tail tip and amplified with primers against the GFP ORF. The sequences were as follows: F-5' TTTCCAAGAAGCTTTAGGGA 3'; R-5' CGAACCAGTTCTACATGCTA 3'. For copy number determination of the Breaker vector, tail tip genomic DNA was amplified with primers against the rtTA ORF. The sequences were as follows: F-5' TCGCGACGGGGCTAAAGTGC 3'; R-5' TGGGGGCATAGAATCGGTGG 3'. To analyze the transcriptional level of the Rosa26 locus, total RNA was extracted from stomach and kidney tissue with an RNAsimple kit (TIAGEN, Cat. #DP419) and reverse transcribed with TransScript ® One-Step gDNA Removal and cDNA Synthesis SuperMix (Trans, Cat. #AT311-02). cDNA was used as a template for real-time PCR with the following primers: F-5' GCCTAAAGAAGAGGCTGTGC 3'; R-5' CGGCTGTCTCACAGAACG 3'. GAPDH was amplified with F-5' ATGACATCAAGAAGGTGGTG 3'; R-5' CATACCAGGAAATGAGCTTG 3' and used as an internal control.

Statistical analysis
GraphPad Prism 8 software was applied for statistical analysis. Sample size was determined on the basis of experience with similar experiments and from that was generally used in other studies. Data were expressed as the mean ± s.d. unless indicated otherwise. Unpaired Student's t-test was employed to determine whether there was a significant difference between two groups. For animal studies, statistical evaluations of in vivo repair    1-year-old IDDoR mice without doxycycline administration were sacrificed for GFP and tdTomato staining. There was no GFP and tdTomato fluorescence observed in the seven organs. The stomach from doxycyclinetreated mice was used as positive control. Scale bar: 20 μm. The NHEJ preference was calculated as GFP + cells% / (GFP + cells% + tdTomato + cells%), and the HR preference was calculated as tdTomato + cells% / (GFP + cells% + tdTomato + cells%). Data were normalized to control mice. The percentage of unrepaired break was calculated as 100% -(GFPMS + cells% + tdTomatoMS + cells%) / (GFPCtrl + cells% + tdTomatoCtrl + cells%). Experiments were repeated at least three times. Ctrl, control mice; MS, mice with mental stress. Error bars represent the s.e.m. The Mann-Whitney U test was employed for significance determination.