Sanger sequencing

CR3022Gl, CR3022Ma and CC12.1 KI mice were generated following published protocols (Lin et al., 2018 (link); Wang et al., 2021a (link)). In brief, the targeting vector 4E10 (Ota et al., 2013 (link)) was modified by the incorporation of human rearranged VDJ (heavy chain construct) or VJ (light chain construct) sequences downstream of the promoter region and by elongation of the 5’ and 3’ homology regions using the Gibson assembly method (NEB). The targeting vector DNA was confirmed by Sanger sequencing (Eton Bioscience Inc.). Next, fertilized mouse oocytes were microinjected with a donor plasmid containing either the pre-rearranged IGH with the mouse V_HJ558 promoter, or the pre-rearranged IGK with the mouse Vκ4-53 promoter (200 ng/μl); two pair of single-guided RNAs (sgRNAs, 25 ng/μl) targeting either the H or the κ locus; and AltR-Cas9 protein (50 ng/μl) and injection buffer (Wang et al., 2021a (link)). Following culture, resulting zygotes were implanted into the uteri of pseudopregnant surrogate C57BL/6J mothers.

PCT64^LMCA KI mice were generated following published protocols (Lin et al., 2018 (link); Wang et al., 2021 (link)). In brief, the targeting vector 4E10 (Ota et al., 2013 (link)) was modified by the incorporation of human rearranged PCT64 LMCA VDJ (heavy chain construct) or VJ (light chain construct) sequences downstream of the promoter region and by elongation of the 5′ AND-3′ homology regions using the Gibson assembly method (NEB). The targeting vector DNA was confirmed by Sanger sequencing (Eton Bioscience Inc.).
Next, fertilized mouse oocytes were microinjected with a donor plasmid containing either the pre-rearranged PCT64 LMCA IGH with the mouse VHJ558 promoter, or the pre-rearranged PCT64^LMCA IGK with the mouse Vκ4-53 promoter (200 ng/μL); two pair of single-guided RNAs (sgRNAs, 25 ng/μL) targeting either the H or the κ locus; and AltR-Cas9 protein (50 ng/μL) and injection buffer (Wang et al., 2021 (link)). Following culture, resulting zygotes were implanted into the uteri of pseudopregnant surrogate C57BL/6J mothers.

Genomic DNA was extracted from fully expanded leaves of Meiwa kumquat using NucleoSpin Plant II (TaKaRa Bio Inc. Kusatsu, Japan). Four fragments, including one fragment covering a 3,456 bp (NCBI accession num’ MT247386) containing the LOB1 promoter, coding region and the PthA4 EBE sequences; one fragment covering 1,264 bp (NCBI accession num’ MT247387) containing the LOB2 promoter and coding regions; and one fragment covering 1,515 bp (NCBI accession num’ MT655137) containing the LOB3 promoter and coding regions, were amplified (primers are shown in S2 Table) from genomic DNA using Q5 High-Fidelity DNA Polymerase (NEB, Ipswich, MA). The LOB1, LOB2 and LOB3 fragments were cloned into pGEM-T (Promega, Madison, WI) or pHSG298 (TaKaRa Bio Inc. Kusatsu, Japan) vectors. Sequences were determined using Sanger sequencing (Eton Bioscience, Inc., San Diego, CA).

Previously described pACYC177 vector plasmids containing ZIKV PRVABC59 genome from either the 5′ UTR to nt 3498 (p1-ZIKV) or from nt 3109 to the end of the 3′ UTR (p2-ZIKV) were used to generate WT ZIKV or the X1 mutant. Primers ZIKV X1 C35G F (5′-TCCCCAAGCTGTGCCTGACTAGCAGGC-3′) and ZIKV X1 C35G R (5′-GCCTGCTAGTCAGGCACAGCTTGGGGA-3′) were used with a QuikChange II XL Site-Directed Mutagenesis Kit (Agilent; Santa Clara, CA) to introduce the X1 C10415G mutation into the p2-ZIKV plasmid. The resulting X1 mutant p2-ZIKV, untreated WT p2-ZIKV, and p1-ZIKV plasmids were rescued and amplified via rolling circle amplification as described [13 (link)]. Prior to in vitro transcription of the viral genomes, the presence of the C10415G mutation in the X1 mutant was confirmed via Sanger sequencing (Eton Bioscience: San Diego, CA, USA).