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Fig. 2 | Journal of Biological Engineering

Fig. 2

From: Viral Cre-LoxP tools aid genome engineering in mammalian cells

Fig. 2

Strategy for establishing TALEN-engineered HEK293 reporter for testing Cre function. a The schematic flowchart shows the unmodified HEK293 cells harboring the wild-type MIR-21 gene at both loci (upper panel). Introduction of TALEN and HR donor DNA results in bi-allelic ablation of MIR-21 [28]. Genome sequence confirmed an indel of 5 bp at one allele and a knockin (KI) and knockout (KO) event at the other allele via homologous recombination (middle panel). These TALEN-engineered HEK293 (HEK293-TE) cells may serve as a reporter cell line for functional test of the CRE activities (lower panel). b The lenti-Cre-GFP vector is introduced into the HEK293-TE cells by transfection. Green fluorescence shows the expression of Cre-GFP (upper left) and red fluorescence shows the expression of floxed-RFP-Puro (upper right). The overlay shows that the Cre-GFP expressing cells do not overlap with the Floxed-RFP-Puro cells(lower left). c Genotyping of the HEK293-TE cells after Cre-GFP introduction. The upper ~430 bp is the indel allele and lower ~350 bp is corresponding to the smaller PCR product resulting from the removal of the floxed reporter cassette. The Foxed reporter cassette (3.1 kb) allele could not be amplified under our experimental conditions. White arrows show GFP and RFP positivity are mutually exclusive. Scale bar 50 μm

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