Construction And Evaluation Of The Ribosomal DNA Targeting Vectors Containing The Recognition Site For ZFNs

Our previous research had suggested that ribosomal DNA (rDNA) locus might serve as a safe and effective harbor for transgene and a non-viral vector system-ribosomal DNA targeting vectors (pHrneo) had been developed, which can target various therapeutic genes into many kinds of human cell lines. By improving the targeting frequencies, rDNA locus may play a more important role in ex vivo cell-based gene therapy, especially in gene targeting of primary cells. Zinc finger nuclease has been widely used as a powerful tool to improve the targeting frequencies, even up to 5000 times. As such, we designed a zinc finger nuclease ZFN-S3 with recognition sites at rDNA+6523-+6546 that proved to have high activity and may be useful for improving targeting frequency of the rDNA locus.Objective:To construct and evaluate the novel rDNA targeting vectors aiming at rDNA ITS 1 locus and containing the recognition sites of ZFN-S3 to improve targeting frequency of the rDNA locus.Methods:Construction of the vector pHrl-NL:firstly, construction of a 2 kb sequence for homologous recombination into the ribosomal DNA locus by PCR amplification and fragment assembling; secondly, insertion of the neomycin phosphotransferase (Neo) gene into the homologous arms to use the promoter-trap strategy for the enrichment of recombinants; finally, adding of two loxP sites on both sides of the selection gene.Construction of the vector pHr2-NL:substitution of rDNA +5513-+6546 sequence for homologous recombination into pHrl-NL vector for rDNA+937-+6546 sequence. pHrl-NL/pHr2-NL plasmid was transferred to human fibrosarcoma cells (HT1080) by nuclear transfection and evaluated by clone screening, picking and counting, as well as PCR analysis.Results:1, The restriction enzyme fragment length of pHrl-NL vector conformed to theoretical value.2, The restriction enzyme fragment length of pHr2-NL vector conformed to theoretical value.3, The sequences of pHrl-NL and pHr2-NL vectors conformed to theoretical sequences.4, After nuclear transfection of pHrl-NL, we did not identify homologous recombinant from 13 resistant cell clones by PCR analysis.5, After nuclear transfection of pHr2-NL, we identified one homologous recombinant from 12 resistant cell clones by PCR analysis.Conclusion:The Neo gene can be successfully targeted into hrDNA ITS1 locus of HT 1080 by the rDNA targeting vector pHr2-NL containing the ZFN recognition sites and two loxP locus, which lays the foundation for high-frequency targeting of the rDNA locus targeting vector combined with ZFN-S3.