Figure 2

Site-specific recombinase-based integration and excision in primary isolated bovine fetal fibroblasts.

(A) Schematic of the pCAG-attBrP2ATK construct and intermediates in pseudo-attP-site integration and Cre/Dre-mediated excision. AttBrP2ATK fusion gene is ubiquitously expressed under the control of the CAGGS promoter until phiC31-mediated integration. EF1a-EGFP is used for visual fluorescence monitoring of the stably-transfected primary cells before Dre-mediated excision and indicates the unexcised transgenic cells after excision. “GOI” represents multiple cloning sites where any DNA sequence can be inserted. TK probe and neo-probe are used for Southern blot analysis to detect Dre- and Cre- mediated excision, respectively. Two arrows that point to opposite directions represent primer pairs used to verify the excision of both selectable makers and plasmid backbone. (B) Relative real-time RT-PCR analysis of EGFP mRNA expression was performed in stably transfected bovine fetal fibroblast cells. The decline of mRNA from 2 to 30 days was not significant (P > 0.05). Error bars denote SEM. (B) Flow cytometry analysis of the percentage of GFP-positive cells was performed in stably transfected bovine fetal fibroblast cells. The decline of percentage of GFP+ cells from 2 to 30 days was not significant (P > 0.05). Error bars denote SEM. (D) Fluorescence phenotype of transgenic cells before and after excision. Scale bar = 20 μm. Approximately 95% of unexcised cells were GFP+ and almost all of the excised cells were GFP negative after FACS sorting. (E) Southern blot analysis of transgenic cell lines before and after Cre/Dre-mediated excision. The single-copy safe harbor integrated clones (#1, #10 and #19) showed a single band of 5.9 kb by using a TK probe or a single band of 8.5 kb by using neo-probe, as depicted by arrow. The unexcised clones (#1, #10, #19, #35, #44 and # 51) carried at least one copy of TK gene or neo-gene, which was no longer detectable after excision (#1e, #10e, #19e, #35e, #44e and # 51e). Full-length blots are presented in Supplementary Figure S9. (F) Cre- and Dre-mediated excision was further demonstrated by PCR. The genomic DNA of untransfected bovine fetal fibroblast cells was used as a negative control. Only the excised cells showed the expected band (334 bp). The gels have been run under the same experimental conditions and full-length gels are presented in Supplementary Figure S10. (G) Panel F, sequencing result of the specific band. The selectable markers and plasmid bacterial backbone were completely removed from the excised cells; rox- and loxP-flanked MCS was retained.