Fig. 4

Transgene Expression from the weak heterologous promoter is principally locus-dependent. CRISPR-mediated integration of ∆CMV-driven EGFP and promoter-less DsRed2 in chicken predicted GSH loci and non-GSH locus was performed in DF1 cell lines. A Schematic depiction of ∆CMV-EGFP-expressing heterogeneous cell pools at the end of MTH2; B Schematic illustration of CMV and ∆CMV promoter as well as negatively- and positively-regulated transcription factor response elements (TFREs). C-a, D-a, E-a) CRISPR-mediated integration of DsRed2-∆CMV-EGFP in cROSA, cHIPP, and cOVA loci verified by 5’/3’ junction PCR, restriction enzyme digestion of the amplicons, and Sanger sequencing; C-b, D-b, E-b) flowcytometry results from EGFP-expressing cROSA, cHIPP, and cOVA cells have been achieved in three individual experiments (each in triplicates). Non-transfected cells have been used as a negative control. Average expression of EGFP for cROSA, cHIPP, and cOVA cell pools has been shown (green square). Expression of EGFP has been detected in the green channel. No expression signal has been detected in the red channel (red square); C–c, C-d, C-e, D-c, D-d, D-e) comparison of integrated density (ID) index, mean fluorescent intensity (MFI) index, and copy numbers (CN) of EGFP transcripts have been conducted among the main experimental groups (i.e., cROSA and cHIPP) against the control group (cOVA). C-f, D-f, E-c, C-g, D-g, E-d) Fluorescence microscope images of the cells expressing ∆CMV-driven EGFP and CMV-driven EGFP heterogeneously (Scale bar: 100um). ns: non-significant, ***: p < 0.005, and ****: p < 0.0001 are statistically significant. Avg.: The average expression of EGFP. N: Number