Abstract
Zinc Finger Nuclease (ZFN) technology has provided researchers with a tool for integrating exogenous sequences into most cell lines or genomes in a precise manner. Using current methods, the efficiency of targeted integration (TI) into the host genome is generally low and is highly dependent on the ZFN activity at the genomic locus of interest. It is unknown if the ZFN binding and cutting efficiency is more dependent on the nucleotide recognition sequence or the chromosomal context in which the sequence is located. We have taken a highly efficient ZFN pair (hAAVS1) from human studies and introduced the exogenous DNA sequence into the Chinese Hamster Ovary (CHO) genome in an attempt to improve the efficiency of targeted integration. A Pad comprised of human AAVS1 sequence has been integrated into the CHO genome at 3 separate loci to determine if the ZFN's will work across species and if the cutting efficiency is affected by chromosomal context. The results of this study will help us to improve the overall efficiency of TI by using Landing Pads, particularly for genomic targets in which suitable ZFN's may not be available.
Highlights
Zinc Finger Nuclease (ZFN) technology has provided researchers with a tool for integrating exogenous sequences into most cell lines or genomes in a precise manner
A “Landing Pad” comprised of human AAVS1 sequence has been integrated into the Chinese Hamster Ovary (CHO) genome at 3 separate loci to determine if the ZFN’s will work across species and if the cutting efficiency is affected by chromosomal context
A plasmid donor carrying the hAAVS1 ZFN recognition sequence Landing Pad was introduced into CHO Rosa26, Site #1, and Neu3 via targeted integration (Figure 1)
Summary
Zinc Finger Nuclease (ZFN) technology has provided researchers with a tool for integrating exogenous sequences into most cell lines or genomes in a precise manner. The efficiency of targeted integration (TI) into the host genome is generally low and is highly dependent on the ZFN activity at the genomic locus of interest. It is unknown if the ZFN binding and cutting efficiency is more dependent on the nucleotide recognition sequence or the chromosomal context in which the sequence is located. We have taken a highly efficient ZFN pair (hAAVS1) from human studies and introduced the exogenous DNA sequence into the Chinese Hamster Ovary (CHO) genome in an attempt to improve the efficiency of targeted integration. The results of this study will help us to improve the overall efficiency of TI by using Landing Pads, for genomic targets in which suitable ZFN’s may not be available
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