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. 2018 Oct 5;19(10):3035. doi: 10.3390/ijms19103035

Figure 2.

Figure 2

The structure of the non-viral vector pMrnF9 and the schematic diagram of gene targeting into the mouse rDNA locus. LHA, long homologous arm, is homologous to the 2527–5646 region of mouse 45S pre-ribosomal DNA sequence. SHA, short homologous arm, is homologous to the 5646–6875 region. Two short flippase recognition target (FRT) sites flank the Neo expression cassette, which links to the internal ribosome entry site (IRES) of encephalomyocarditis virus. By promoter trapping, Neo can be transcribed via the in situ rDNA promoter after homologous recombination. A universal promoter EF1-α is utilized to drive the human F9 expression cassette. Positive targeted cells with site-specific integration of exogenous genes can be filtered by PCR using primer F1 and R1 with a product of 1679 bp. When the genome DNA of targeted clones is digested by PvuIIand detected with the probe homologous to the IRES element, the site-specific clones will show a band of 6208 bp. The Neo cassette can be excised by expression of Flpe recombinase. The excised clones can be filtrated by PCR with the primer F2 and R2. A 463 bp band will appear from excised clones while a 2178 bp band remains in original clones.