PCT64LMCA 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 PCT64LMCA 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.
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 PCT64LMCA 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.
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