Supplementary Figure 1: Cre-loxP and CRISPR–Cas9 approaches to generate HS-mutant MLEC lines.
From: A mutant-cell library for systematic analysis of heparan sulfate structure–function relationships
The primary MLECs were isolated from mice with HS genes systematically or conditionally targeted (loxP sites flanking the target gene, indicated by “floxed” or “f”). The primary MLECs were immortalized by expression of SV40 T antigen and then single-cell cloned to obtain MLEC lines. Next, the floxed gene was deleted by transient expression of Cre recombinase to derive the ‘daughter’ HS-mutant cell line. In addition, a ‘wild-type’ floxed (Ndst1f/f) MLEC line was cotransfected with target-gene-specific gRNA and Cas9 and then screened for the targeted gene insertion/deletion (indel) mutation and subjected to cell cloning to obtain HS-mutant cell lines. PAM, protospacer-adjacent motif.
