Targeted gene knockout in mammalian cells by using engineered zinc-finger nucleases

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   Fig. 1.

   Targeting the DHFR locus with designed ZFNs. (A) Section of the DHFR gene targeted by ZFNs. The DNA sequence of the primary binding site for each ZFN is boxed. ZFN7843 and ZFN9461 bind the same 12-bp site. ZFN7844 binds the 12-bp site AATGCTCAGGTA, whereas ZFN9684 binds the 15-bp site AATGCTCAGGTACTG. (B) Recognition helix sequences of ZFNs. The sequence of the recognition helix from position −1 to +6 (27) is listed below its target triplet. Backbone sequences for the ZFPs can be found elsewhere (20). The C-terminal finger recognizes the 5′ most DNA triplet. The asterisks denote that these ZFNs employ the ELKK variant FokI domains throughout. (C) Comparison of ZFN activity. Plasmids encoding each pair of ZFNs (ZFN7843/ZFN7844 and ZFN9461/ZFN9684) containing the ELKK FokI variants were delivered in the amounts shown to CHO-S cells in suspension culture. The frequency of allelic mutation in each pool of treated cells was determined by using the CEL-I assay (gel). Bands migrating at 384, 204, and 180 bp represent the parent amplicon and the two CEL-I digestion products, respectively. The bands were quantitated by EtBr staining and densitometry to determine the frequency of NHEJ. The frequency of NHEJ is plotted against ZFN dosage. The lowest band on the 25-bp size ladder is 125 bp.

   
   
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   Fig. 2.

   Genotype and phenotype of ZFN-induced DHFR ^−/− clones from CHO-S cells. (A) Each pair of sequences represents the two alleles of the DHFR gene in the designated cell line. For each mutant allele, inserted bases are boxed, and deleted bases are represented by dots. (B) Western blot for DHFR protein in WT CHO-S cells (WT) and the mutant cell lines 14/1, 14/2, and 1.43 and the commercially available DHFR-null CHO cell line DG44. Note that, for clone 1.43, we analyzed three independent subclones from this line (H7, B11, and A5). Input is normalized against TFIIB or glutamine synthetase (GS) expression levels as indicated. (C) Fluorescein-labeled methotrexate (FMTX)-based FACS analysis of DHFR ^−/− cell lines. WT(-ve), WT CHO-S cells not treated with FMTX; WT(+ve), WT CHO-S cells treated with FMTX. All mutant cell lines were treated with FMTX, including DG44 as a DHFR-null CHO cell control. (Left) Cells in adherent culture. (Right) Cells in suspension culture.

   
   
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   Fig. 3.

   Growth and functional properties of the ZFN-generated DHFR ^−/− cell lines. (A) WT CHO-S (WT CHO) and ZFN-generated DHFR ^−/− CHO-S cell lines were cultured in the presence or absence of HT supplement as indicated. (Upper) Growth of DHFR ^−/− cell lines 14/1 and 14/2 compared with WT CHO cells in adherent culture conditions. (Lower) Growth and viability of the DHFR ^−/− cell line 1.43A5 in serum-free suspension culture. (B) IgG expression from WT or DHFR ^−/− cell lines after transfection with antibody expression constructs coexpressing a DHFR gene as a selection marker. Cells were cultured in the presence (+HT) or absence (−HT) of HT supplement for 14 days. IgG expression at >48 h was measured by ELISA. WT Ad., WT CHO-S cells in adherent medium; WT Susp., WT CHO-S cells in suspension medium. Cell lines 14/1 and 14/2 were cultured adherently. Cell line 1.43A5 was cultured in suspension. (C) Methotrexate selection/amplification in DHFR ^−/− cell line 14/1. Data shown is the level of IgG stably expressed from a clone 14/1 pool after 2 weeks of incubation in the absence of HT then 2 weeks more in the presence of the noted concentration of methotrexate (see Materials and Methods).