Journal: iScience

Article Title: Efficient genome editing in erythroid cells unveils novel MYB target genes and regulatory functions

doi: 10.1016/j.isci.2023.107641

Figure Lengend Snippet: Knock-in efficiency (A) The KI efficiencies using the indicated targeting vector in the various CAS9-expressing MEL cells are shown as percentage. The number of KI clones over the total number of screened clones is indicated between brackets. (B) PCR screening of sorted GFP+ MEL cell clones. Top lane: KI-specific PCR product, bottom lane: genomic DNA control PCR. The Myb -GFP KI clones are labeled in green on top of the gel. See also Figures S2–S5 .

Article Snippet: Viral plasmids expressing Cas9: pLentiCRISPRv2_Neo-short was obtained by deleting the Acc65I/EcoRI fragment from the pLentiCRISPRv2_Neo vector (Addgene 98292), and TLCV2-T2A-noGFP-puro by deleting the BamHI/NheI GFP fragment from TLCV2 (Addgene 87360).

Techniques: Knock-In, Plasmid Preparation, Expressing, Clone Assay, Control, Labeling

Journal: iScience

Article Title: Efficient genome editing in erythroid cells unveils novel MYB target genes and regulatory functions

doi: 10.1016/j.isci.2023.107641

Figure Lengend Snippet: Cellular and molecular characterization of Myb -GFP and Zeb1 -GFP KI MEL cells (A) FACS analysis of heterozygous ( Myb -GFP GFP/WT ) and homozygous ( Myb -GFP GFP/GFP ) KI cells obtained from the different CAS9-expressing parental MEL cells. The MFI are indicated in each plot. (B) Western blotting analysis of Myb -GFP KI MEL cells using anti-GFP, and anti-MYB antibodies. Anti-GAPDH was used as loading control. (C) Two different PCR genotyping of Zeb1-GFP KI cells are shown, highlighting the presence of the endogenous Zeb1 (945 bp) signal in the control and heterozygous clones, but not in the homozygous KI cells. Specific amplification of the KI allele is also shown, which is uniquely found in the KI cells (bottom panel, 1,092 bp). (D) FACS analysis of Zeb1-GFP KI cells. The heterozygous (blue) and homozygous (green) MFI values are indicated. (E) Western blotting analysis of the Zeb1-GFP KI cells using an anti-ZEB1 antibody, highlighting endogenous ZEB1 and ZEB1-GFP proteins. See also Figure S6 .

Article Snippet: Viral plasmids expressing Cas9: pLentiCRISPRv2_Neo-short was obtained by deleting the Acc65I/EcoRI fragment from the pLentiCRISPRv2_Neo vector (Addgene 98292), and TLCV2-T2A-noGFP-puro by deleting the BamHI/NheI GFP fragment from TLCV2 (Addgene 87360).

Techniques: Expressing, Western Blot, Control, Clone Assay, Amplification

Journal: iScience

Article Title: Efficient genome editing in erythroid cells unveils novel MYB target genes and regulatory functions

doi: 10.1016/j.isci.2023.107641

Figure Lengend Snippet: Functional characterization of Myb -GFP KI MEL cells (A) Growth curves of MEL cells, MEL-CAS9, and two MEL Myb -GFP clones: 1 homozygous (hom), and 1 heterozygous (het). (B) Expression of the Epb4.2 , Gypa , and Myb genes upon induction of differentiation. (C) Mean Fluorescence intensity (MFI) of heterozygous and homozygous MEL Myb -GFP cells showing the rapid drop of GFP as a function of differentiation time. Note that homozygous KI cells show increased MFI compared to heterozygous cells, as expected. Data are represented as mean ± SEM.

Article Snippet: Viral plasmids expressing Cas9: pLentiCRISPRv2_Neo-short was obtained by deleting the Acc65I/EcoRI fragment from the pLentiCRISPRv2_Neo vector (Addgene 98292), and TLCV2-T2A-noGFP-puro by deleting the BamHI/NheI GFP fragment from TLCV2 (Addgene 87360).

Techniques: Functional Assay, Clone Assay, Expressing, Fluorescence