Snapgene 6

Mutagenic PCR primers were designed in silico to introduce the identified mutations using SnapGene 6.0.2 software (GSL Biotech). We performed PCRs using the SuperFi II Master Mix (Thermo Fisher 12368010), and the products were gel purified using the Machary-Nagel NucleoSpin Gel and PCR Clean-up kit (740609). We assembled the products using the NEB Builder HiFi DNA Assembly Master Mix (E2621L). The assemblies were digested with DpnI (R0176S), Lambda exonuclease (M0262S), and Exonuclease I (M0293S) and amplified using SuperPhi RCA Premix Kit with Random Primers (Evomic Science catalog number PM100) (Bates et al., 2020 (link)). As previously described, RCA products were then transfected into HEK293A cells to produce p0 stocks of the virus (Marano et al., 2020 (link); Marano et al., 2022 ). The p0 stocks were then used to infect Vero cells at an MOI of 0.01 to produce p1 stocks, which were used for all downstream tests. We sequenced the p1stocks of the mutant viruses derived from the anti-DENV passaged population using both Sanger sequencing and next-generation sequencing to confirm the mutation of interest was introduced. The p1 stocks of the mutant viruses derived from the control passaged population were sequenced by Sanger sequencing to confirm the mutation was properly introduced.

Genome DNA was extracted from the tail tip of 1-week-old F0 mice. The offspring genotypes were identified via PCR with forward and reverse primers (Table 1). After purification, PCR products were sequenced with Sanger sequencing and the results were analyzed with SnapGene 6.0.2 software (GSL Biotech, Chicago, IL, USA).
PCR-confirmed founder mice were mated with wildtype NOD mice to generate F1; positive F1 animals derived from same genotype founder mice were intercrossed to produce F2. Heterozygous F2 animals were intercrossed until obtaining homozygous mice (dclre1c-NOD mice).