Zinc-finger nucleases (ZFNs) have enabled highly efficient gene targeting in multiple cell types and organisms. Here we describe methods for using simple ssDNA oligonucleotides in tandem with ZFNs to efficiently produce human cell lines with three distinct genetic outcomes: (i) targeted point mutation, (ii) targeted genomic deletion of up to 100 kb and (iii) targeted insertion of small genetic elements concomitant with large genomic deletions.

The nuclear receptor peroxisome proliferator-activated receptor ␥ (PPAR␥) promotes adipocyte differentiation, exerts atherogenic and anti-inflammatory effects in monocyte/macrophages, and is believed to mediate the insulin-sensitizing action of antidiabetic thiazolidinedione ligands. As no complete PPAR␥ antagonists have been described hitherto, we have constructed a dominant-negative mutant receptor to inhibit wild-type PPAR␥ action. Highly conserved hydrophobic and charged residues (Leu 468 and Glu 471 ) in helix 12 of the ligand-binding domain were mutated to alanine. This compound PPAR␥ mutant retains ligand and DNA binding, but exhibits markedly reduced transactivation due to impaired coactivator (cAMP-response elementbinding protein-binding protein and steroid receptor coactivator-1) recruitment. Unexpectedly, the mutant receptor silences basal gene transcription, recruits corepressors (the silencing mediator of retinoid and thyroid receptors and the nuclear corepressor) more avidly than wild-type PPAR␥, and exhibits delayed ligand-dependent corepressor release. It is a powerful dominant-negative inhibitor of cotransfected wild-type receptor action. Furthermore, when expressed in primary human preadipocytes using a recombinant adenovirus, this PPAR␥ mutant blocks thiazolidinedione-induced differentiation, providing direct evidence that PPAR␥ mediates adipogenesis. Our observations suggest that, as in other mutant nuclear receptor contexts (acute promyelocytic leukemia, resistance to thyroid hormone), dominant-negative inhibition by PPAR␥ is linked to aberrant corepressor interaction. Adenoviral expression of this mutant receptor is a valuable means to antagonize PPAR␥ signaling.Peroxisome proliferator-activated receptor ␥ (PPAR␥), 1 an orphan member of the nuclear hormone receptor family, was first characterized as a transcription factor that regulates adipocyte-specific gene expression (1) and induces preadipocyte differentiation (2), but is now recognized to have a central role in other biological processes. PPAR␥ mediates inhibition of inflammatory cytokine production (interleukin-6 and tumor necrosis factor ␣) from monocytes (3), and receptor activation by oxidized low density lipoprotein-derived ligands promotes macrophage foam cell formation (4). PPAR␥ activation promotes colonic neoplasia (5), but inhibits the growth of breast cancer cells (6). Thiazolidinediones (TZDs), a novel class of antidiabetic agent that act as insulin sensitizers in vivo, bind PPAR␥ with high affinity (7), and prostaglandin J2 (8) and fatty acids have been proposed to be natural ligands. PPAR␥ regulates target gene transcription as a heterodimer with the retinoid X receptor, and this heterodimeric complex has been shown to be activated synergistically by TZDs and RXR-specific ligands (9). However, no complete synthetic or natural PPAR␥ antagonists have been described hitherto. We have therefore generated a dominant-negative PPAR␥ mutant to inhibit wildtype receptor action.In keeping with other members of the nuclear receptor...

Gene knockout is the most powerful tool for determining gene function or permanently modifying the phenotypic characteristics of a cell. Existing methods for gene disruption are limited by their efficiency, time to completion, and/or the potential for confounding off-target effects. Here, we demonstrate a rapid single-step approach to targeted gene knockout in mammalian cells, using engineered zinc-finger nucleases (ZFNs). ZFNs can be designed to target a chosen locus with high specificity. Upon transient expression of these nucleases the target gene is first cleaved by the ZFNs and then repaired by a natural-but imperfect-DNA repair process, nonhomologous end joining. This often results in the generation of mutant (null) alleles. As proof of concept for this approach we designed ZFNs to target the dihydrofolate reductase (DHFR) gene in a Chinese hamster ovary (CHO) cell line. We observed biallelic gene disruption at frequencies >1%, thus obviating the need for selection markers. Three new genetically distinct DHFR ؊/؊ cell lines were generated. Each new line exhibited growth and functional properties consistent with the specific knockout of the DHFR gene. Importantly, target gene disruption is complete within 2-3 days of transient ZFN delivery, thus enabling the isolation of the resultant DHFR ؊/؊ cell lines within 1 month. These data demonstrate further the utility of ZFNs for rapid mammalian cell line engineering and establish a new method for gene knockout with application to reverse genetics, functional genomics, drug discovery, and therapeutic recombinant protein production.genetic engineering ͉ zinc-finger proteins T he use of gene knockouts in basic research, functional genomics, and industrial cell line engineering is severely limited by an absence of methods for rapid targeting and disruption of an investigator-specified gene. Early approaches to somatic cell gene disruption used genome-wide nontargeted methods, including ionizing radiation and chemical-induced mutagenesis (1, 2) whereas more recent methods used targeted homologous recombination (HR) (3). However, the Ͼ1,000-fold lower frequency of the targeted HR event relative to random integration in most mammalian cell lines (beyond mouse ES cells) can necessitate screening thousands of clones and take several months to identify a biallelic targeted gene knockout. Strategies including positive and negative marker selection and promoter-trap can boost efficiencies considerably, although these approaches present their own technical challenges and are not always successful in achieving high efficiency targeting (4, 5). Although advances with adeno-associated viral delivery strategies continue to improve the efficiency of knockouts (6, 7), the frequency is still very low and the time required to achieve biallelic gene knockout remains a barrier to its routine adoption. Here, we present a general solution for rapid gene knockout in mammalian cells.The repair of double strand DNA breaks (DSB) in mammalian cells occurs via the distinct mechanisms of homol...

To determine functional differences between the two splice variants of PPAR␥ (␥1 and ␥2), we sought to selectively repress ␥2 expression by targeting engineered zinc finger repressor proteins (ZFPs) to the ␥2-specific promoter, P2. In 3T3-L1 cells, expression of ZFP55 resulted in >50% reduction in ␥2 expression but had no effect on ␥1, whereas adipogenesis was similarly reduced by 50%. However, ZFP54 virtually abolished both ␥2 and ␥1 expression, and completely blocked adipogenesis. Overexpression of exogenous ␥2 in the ZFP54-expressing cells completely restored adipogenesis, whereas overexpression of ␥1 had no effect. This finding clearly identifies a unique role for the PPAR␥2 isoform. The nuclear hormone receptor PPAR␥ is essential for cellular differentiation and lipid accumulation during adipogenesis (Barak et al. 1999;Kubota et al. 1999;Rosen et al. 1999). The adipocyte-specific ␥2 isoform differs from the more widely expressed ␥1 in that it contains additionally 30 amino acid residues at the amino terminus (Kliewer et al. 1994;Tontonoz et al. 1994a;Zhu et al. 1995). Evidence suggests these residues contribute to a constitutive transcription activation function that is 5-10-fold greater than in ␥1 (Werman et al. 1997). PPAR␥2 is selectively expressed in adipose tissue (Fajas et al. 1997) and is strongly up-regulated during adipogenesis (Tontonoz et al. 1994b;Wu et al. 1998), suggesting a specific role for this isoform in fat cell differentiation. Nevertheless, a specific role for ␥2 that could not be substituted by ␥1 has not been clearly determined.The ability to selectively knock out or knock down the expression of a specific gene provides a powerful approach for understanding its biological function. The targeting of individual mRNA splice variants offers an even greater level of selective control and understanding of differential isoform function. Rationally engineered transcription factors potentially provide a powerful tool for targeted regulation of endogenous genes by combining a functional transcription regulatory domain with a customized DNA binding domain that can bind to a specific sequence within the target gene. C2H2 zinc finger proteins (ZFPs) can be engineered to bind with high specificity to wide a diversity of DNA sequences (Desjarlais and Berg 1992;Choo and Klug 1994;Jamieson et al. 1994;Rebar and Pabo 1994;Greisman and Pabo 1997). Previous studies have demonstrated the utility of both engineered activator-and repressor-ZFPs in the regulation of endogenous chromosomal loci (Bartsevich and Juliano 2000;Beerli et al. 2000;Zhang et al. 2000;Liu et al. 2001). Our goal for this study was to selectively inhibit expression of the PPAR␥2 isoform in the adipogenic mouse 3T3-L1 cell line by utilizing engineered zinc finger repressor proteins. Results and DiscussionThe mouse PPAR␥ gene spans >105 kb (Zhu et al. 1995). Coding exons 1 to 6 are conserved between the ␥1 and ␥2 isoforms (Fig. 1A) and transcription of these is driven by an upstream promoter (P1) that also drives expression of two untrans...