Figure 1.
Loss of Pdap1 in CH12 cells impairs CSR.(A) Scheme of murine Pdap1 genomic locus and location of gRNAs used for gene targeting (scheme adapted from Ensembl Pdap1-201 ENSMUST00000031627.8). (B) Top left: Representative flow cytometry plots measuring CSR to IgA in activated Cas9/gPdap1-nucleofected CH12 cells. Numbers in the plots refer to the percentage of switched cells (= IgA+ cells). Top right: Summary graph for three independent experiments using gPdap1-1/3 (individually or in pooled format) or Nickases-a/c (individually). Controls for gRNA-nucleofected CH12 were cells electroporated with either empty vector or gRNAs against random sequences not present in the mouse genome (gCtrl). A previously described gRNA against the CSR factor 53BP1 (Delgado-Benito et al., 2018) was used as a positive control for loss-of-CSR function. Bottom: WB analysis of whole-cell extracts from CH12 cultures nucleofected with Nickase-a to -c. Triangles indicate twofold dilution. Asterisk denotes aspecific bands used for internal normalization of protein levels. N, Nickase.(C) Graph summarizing CSR efficiency of activated CH12 clonal cell lines derived by nucleofection of CH12 cultures with Nickase-a/c and single-cell sorting. Each symbol in the graphs indicates a single-cell clonal derivative. (D) Left: Genomic scars of the targeted alleles in the selected Pdap1-deficient CH12 clonal derivatives. fs, frameshift; PTC, premature termination codon; Δ, bp deletion; het, heterozygous configuration (different indels causing fs and PTC at the two Pdap1 alleles); homo, homozygous configuration. Right: Representative WB analysis of WT and Pdap1-deficient CH12 cell lines. R1 and R2 are WT clonal derivatives generated by targeting CH12 with random sequences not present in the mouse genome. mut, mutated. Asterisk denotes aspecific bands. Numbers underneath the blot indicate relative quantification of Pdap1 signal. (E) Left: Representative flow cytometry plots measuring CSR to IgA in activated CH12 cell lines of the indicated genotypes. WT controls included both the parental CH12 cell line (WT) and the random clonal derivatives R1 and R2. Right: Summary graph for four independent experiments. Significance in B, C, and E was calculated with the Mann–Whitney U test, and error bars represent SD. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

Loss of Pdap1 in CH12 cells impairs CSR. (A) Scheme of murine Pdap1 genomic locus and location of gRNAs used for gene targeting (scheme adapted from Ensembl Pdap1-201 ENSMUST00000031627.8). (B) Top left: Representative flow cytometry plots measuring CSR to IgA in activated Cas9/gPdap1-nucleofected CH12 cells. Numbers in the plots refer to the percentage of switched cells (= IgA+ cells). Top right: Summary graph for three independent experiments using gPdap1-1/3 (individually or in pooled format) or Nickases-a/c (individually). Controls for gRNA-nucleofected CH12 were cells electroporated with either empty vector or gRNAs against random sequences not present in the mouse genome (gCtrl). A previously described gRNA against the CSR factor 53BP1 (Delgado-Benito et al., 2018) was used as a positive control for loss-of-CSR function. Bottom: WB analysis of whole-cell extracts from CH12 cultures nucleofected with Nickase-a to -c. Triangles indicate twofold dilution. Asterisk denotes aspecific bands used for internal normalization of protein levels. N, Nickase.(C) Graph summarizing CSR efficiency of activated CH12 clonal cell lines derived by nucleofection of CH12 cultures with Nickase-a/c and single-cell sorting. Each symbol in the graphs indicates a single-cell clonal derivative. (D) Left: Genomic scars of the targeted alleles in the selected Pdap1-deficient CH12 clonal derivatives. fs, frameshift; PTC, premature termination codon; Δ, bp deletion; het, heterozygous configuration (different indels causing fs and PTC at the two Pdap1 alleles); homo, homozygous configuration. Right: Representative WB analysis of WT and Pdap1-deficient CH12 cell lines. R1 and R2 are WT clonal derivatives generated by targeting CH12 with random sequences not present in the mouse genome. mut, mutated. Asterisk denotes aspecific bands. Numbers underneath the blot indicate relative quantification of Pdap1 signal. (E) Left: Representative flow cytometry plots measuring CSR to IgA in activated CH12 cell lines of the indicated genotypes. WT controls included both the parental CH12 cell line (WT) and the random clonal derivatives R1 and R2. Right: Summary graph for four independent experiments. Significance in B, C, and E was calculated with the Mann–Whitney U test, and error bars represent SD. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal