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. 2002 Jun 15;30(12):e59. doi: 10.1093/nar/gnf059

Figure 5.

Figure 5

NlsCre-mediated recombination in mouse bone marrow (BM) cells. Aliqouts of 2 × 106 BM cells prepared from homozygous C/EBPαfl/fl mice, with both C/EBPα genes floxed (14), were incubated for 7 h in medium containing 1.5 µM nlsCre or PTD-nlsCre. As a negative control, an equivalent volume of PBS was added (mock). Genomic DNA prepared from these cells was used as a template for PCR reactions to confirm the presence of the C/EBPαfl/fl allele (PCR1) and to detect the recombined allele, produced by Cre/lox-specific excision of the C/EBPα gene (PCR2). The 269 bp product produced in PCR1 indicated the C/EBPαfl/fl knock-in allele. A 400 bp fragment, which was generated in the second PCR only when excision of C/EBPα occured, could only be detected when the BM cells were incubated with nlsCre or PTD-nlsCre. Sequencing of the 400 bp product confirmed that exact lox-specific recombination occurred. Therefore, primary bone marrow cells were genetically modified by external addition of nlsCre protein.