Transgenic Res (2010) 19:363–371 DOI 10.1007/s11248-009-9323-7

REVIEW

Zinc-finger : a powerful tool for of animals

Se´verine Re´my Æ Laurent Tesson Æ Se´verine Me´noret Æ Claire Usal Æ Andrew M. Scharenberg Æ Ignacio Anegon

Received: 13 August 2009 / Accepted: 10 September 2009 / Published online: 26 September 2009 Ó Springer Science+Business Media B.V. 2009

Abstract The generation of genetically modified perspective manuscript is a short review on the use animals or plants with -targeted deletions or of ZFNs for the genetic engineering of plants and modifications is a powerful tool to analyze gene animals, with particular emphasis on our recent work function, study disease and produce organisms of involving . We also discuss the application of economical interest. Until recently, the generation of other targeted nucleases, including homing endonu- animals with gene targeted manipulations has been cleases. Microinjection of or mRNA for accomplished by (HR) in ZFNs into embryos allowed targeted, rapid, embryonic stem (ES) cells or cloning through nuclear complete, permanent and heritable disruption of transfer and has been limited to a few species. endogenous loci. The application of ZFNs to generate Recently, a new technology based on the use of gene- gene-targeted knockouts in species where ES cells or targeted zinc-finger nucleases (ZFNs) was developed cloning techniques are not available is an important and used for the generation of organisms with gene- new development to answer fundamental biological targeted deletions and/or modifications when com- questions and develop models of economical interest bined with HR. ZFNs have been used to generate such as for the production of humanized antibodies. modified organisms such as plants, Drosophila, zebra Further refinements of ZFN technology in combina- fish and rats with gene-targeted mutations. This tion with HR may allow knock-ins in early embryos even in species where ES cells or cloning techniques are available. S. Re´my L. Tesson S. Me´noret C. Usal I. Anegon (&) Keywords Zinc finger nucleases INSERM, U643, 44093 Nantes, France Targeted transgenesis Rat e-mail: [email protected] Homologous recombination Animal models S. Re´my L. Tesson S. Me´noret C. Usal I. Anegon CHU Nantes, Institut de Transplantation et de Recherche en Transplantation, ITERT, 44093 Nantes, France

S. Re´my L. Tesson S. Me´noret C. Usal I. Anegon Universite´ de Nantes, Faculte´ de Me´decine, Introduction 44093 Nantes, France Techniques available for modifications are A. M. Scharenberg Seattle Children’s Research Institute, 1900 9th Avenue, either random or targeted. involves HR Seattle, WA 98103, USA using ES cells or nuclear transfer and thus is limited to 123 364 Transgenic Res (2010) 19:363–371 species in which appropriate cells are available, i.e., 2008; Shukla et al. 2009; Szczepek et al. 2007). FokI mice, pigs and cows. Random mutagenesis involves requires dimerization to cut DNA. The binding of two the use of chemicals or transposons. Although these heterodimers of designed ZFN-FokI hybrid mole- techniques have been used for the generation of many cules to two contiguous target sequences in each useful mutants in many different species, they are DNA strand separated by a 6 base-pair cleavage site cumbersome and expensive and do not allow targeted results in FokI dimerization and subsequent DNA modification of specific . Therefore, the gener- cleavage. ation of transgenic organisms with gene-targeted The specificity of ZFN’s is determined by their modifications has been hampered in most species by polymeric zinc finger domains, the DNA binding the lack of appropriate technologies. properties of which are generated through modular The rat is an important biomedical research model assembly of individual zinc fingers [reviewed in whose utility has been significantly hampered by a (Beerli and Barbas 2002; Pabo et al. 2001)]. Two lack of technologies for targeted genome modification major platforms exist for generating polymeric zinc (Aitman et al. 2008; Jacob 2009). Transgenic rats have fingers with defined specificities: a proprietary plat- been generated using microinjection of DNA (Char- form developed by Sangamo Biosciences (Isalan et al. reau et al. 1996;Me´noret et al. 2009; Mullins et al. 2001; Urnov et al. 2005), and the OPEN platform 1990) or lentiviral vectors (Lois et al. 2002; Pfeifer developed by the Consortium (Maeder 2006;Re´my et al. 2009). Over 195 transgenic or et al. 2008; Sander et al. 2007; Wright et al. 2006). Both mutant rats have been produced using chemicals (Zan are now accessible for transgenic animal research et al. 2003) or transposons (Kitada et al. 2007; Tesson purposes: Sangamo has partnered with Sigma to sell et al. 2005) (for a complete list see: http://www.ifr26. pre-assembled ZFN’s via the Compozr program univ-nantes.fr/ITERT/transgenese-rat/liste_rats.php). (http://www.compozrzfn.com/), while the OPEN plat- Rat cloning using nuclear transfer (Cozzi et al. 2009; form (http://www.addgene.org/zfc; www.zincfingers. Zhou et al. 2003) as well as the generation of rat ES org/software-tools.htm) makes their modular assembly cells (Buehr et al. 2008; Li et al. 2008), induced plu- zinc finger pools and reagents freely available. ripotent stem cells (iPS) (Kawamata and Ochiya 2009; Induction of a DNA double strand break by a ZFN Ueda et al. 2008) and spermatogonial stem cells (SSC) results in the activation of a cellular response known (Shinohara et al. 2006) have been described recently, as the DNA damage response. A double strand break but to date the cells used for these procedures have not can be repaired in two different ways (Fig. 1). Non- been demonstrated to be amenable to any form of homologous end joining (NHEJ) generates short targeted gene modification. insertions or deletions at the cleavage site. Repair This perspective is a follow-up to our recent by HR using a DNA template results in gene knock- publication, describing the first generation of knockout ins that are either a perfect repair or, if a modified rats using a newly developed technique that applies template is introduced, sequence replacement. zinc-finger nucleases (ZFNs) in rat embryos (Geurts Zinc-finger nucleases (ZFNs) have been used to et al. 2009a, b). We discuss our approach and the generate gene-targeted knockin in cultured cells present state of the field, and highlight important issues (Bibikova et al. 2003), including human ES and iPS for investigators to consider in applying - cells (Hockemeyer et al. 2009), plants (Cai et al. 2009; mediated gene targeting to animal models. Townsend et al. 2009) and Drosophila (Beumer et al. 2008; Bibikova et al. 2002; Bozas et al. 2009) as well as gene-targeted knockouts in Xenopus, C. elegans Zinc finger nucleases and related technologies (Carroll et al. 2008), and zebra fish (Doyon et al. 2008; for targeted genetic modification Foley et al. 2009). ZF fused to transcription activators have also been used to target specific promoter ZFNs are hybrid molecules composed of a designed sequences and induce gene expression in transgenic polymeric zinc finger domain specific for a DNA mice (Mattei et al. 2007). ZFNs have also been used to target sequence and a FokI nuclease cleavage domain induce gene-targeted knock-ins in cells in the context (Kandavelou and Chandrasegaran 2009; Kim et al. of for (Lombardo et al. 1997; Miller et al. 2007; Porteus 2008; Santiago et al. 2007; Moehle et al. 2007; Perez et al. 2008). Related 123 Transgenic Res (2010) 19:363–371 365

ZFNs

Target sequence (exon of coding gene)

ZFNs

Double strand break

Homologous Repair Non Homologous End-Joining (NHEJ) (Template)

gene disruption in ~70 % of NHEJ Targeted events by out of frame deletions insertion

Fig. 1 ZFN mediated targeted genome modification relies on break by non-homologous end joining, which is imperfect and cellular DNA repair pathways. ZFNs recognize specific gene generates in *70% of the cases gene deletions due to out of sequence in each DNA strand of a given loci and are separated frame deletions of variable lengths, or by homologous by a 6 base pair sequence where the FokI nuclease will recombination when a repair sequence is introduced into the generate a double strand break. The cellular DNA repair the cell together with the ZFNs technologies such as homing endonucleases utilize a Since IgM cell membrane expression on B lympho- different DNA cleavage mechanism, but the resulting cytes is needed for their survival and the expression DNA double strand break is resolved through the same of all the other Ig isotypes (Kitamura et al. 1991) mechanisms and engineered homing endonucleases these IgM-knockout rats should be mature B lym- (Monnat et al. 2008; Paques and Duchateau 2007; phocytes- and heavy chain Ig-deficient. These Ig- Stoddard et al. 2008) or synthetic nucleases (Cannata deficient rats would be useful models for the analysis et al. 2008) can be applied in the same manner as of the role of antibodies in a variety of pathophys- ZFNs. The higher engineering barriers to generating iological situations. Additionally, these Ig-deficient novel homing endonucleases has so far limited their rats could be crossed with transgenic rats for the application to a few plant (Yang et al. 2009) and human heavy and light chain Ig and thus constitute a human applications (Grizot et al. 2009; Paques and platform for the production of human antibodies Duchateau 2007), although recent progress should directed against any target with potential clinical allow more widespread application as the engineering interest. In the same manuscript (Geurts et al. 2009b), barriers to developing novel homing endonucleases the group of H. J. Jacobs generated knockout rats for are reduced (Arnould et al. 2006; Ashworth et al. a GFP transgene inserted into the genome by 2006; Takeuchi et al. 2009; Volna et al. 2007). lentiviral transgenesis and an endogenous gene, Rab38. Mutants were isolated with three different rat inbred or outbred strains. Zinc finger nucleases-mediated targeted gene For our approach to generating gene-targeted rats, modification in rats ZFNs targeted to the IgM locus (IgM ZFNs) were cloned in cis into an expression plasmid as two open We have recently described that expression of ZFNs reading frames linked by the self-cleaving peptide in rat embryos resulted in a high frequency of T2A under the transcriptional control of the CAG mutated live offspring with complete, permanent and promoter and also containing 30 polyA sequences heritable disruptions of the immunoglobulin (Ig) (Fig. 2) (Geurts et al. 2009a, b). The ZFN-encoding heavy chain l locus (IgM) (Geurts et al. 2009a, b). circular plasmid DNA or mRNA were delivered by

123 366 Transgenic Res (2010) 19:363–371 pronuclear or intracytoplasmic injection (Fig. 2)in fidelity for each target sequence as no ZFN-induced order to limit the duration of ZFN expression. ZFNs mutations were detected in target-gene-disrupted were injected at different concentrations and the animals at any of 12 predicted ZFN off-target sites. percentage of mutated embryos (5–75%) obtained Importantly, after breeding to wild-type animals, 1/1 was directly correlated to the concentration of GFP and 5 out of 7 IgM mutant rats transmitted injected ZFN vector [Table 1; (Geurts et al. 2009a, mutated alleles through the germline (Table 2). b)]. Sequence analysis of 22 founders revealed Phenotypic analysis of animals from one of these deletions ranging from 3 to 224 base pairs. Some of IgM- lines showed undetectable or very the animals showed more than one deletion, indicat- low serum levels of IgM, IgG, IgA and IgE (man- ing ZFNs acted not only at the zygote stage but also uscript in preparation), confirming that the deletion in at later time points of embryonic development. While the IgM CH1 region resulted in Ig deficiency and not most of the founders showed one normal IgM allele, in alternative splicing and generation of Ig of shorter one animal carried biallelic mutations in IgM. ZFN- l heavy chain length, as previously reported in mice mediated gene disruption also demonstrated high (Zou et al. 2001). Taken together, our application of plasmid or Rat one-cell embryo mRNA ZFN-mediated gene inactivation in rats ZFN1-ZFN2 resulted in a single-step whole rat gene knockout •DNA expression vector •mRNA (capped and polyA) that was targeted, rapid, complete and permanent. ZFNs were expressed transiently and showed mini- CAGp ZFN 1-2A-ZFN 2 pA mal off-target effects in both inbred and outbred rat strains. These Ig-deficient rats should be a useful biomedical research model. Further work is ongoing to knockout the rat Ig J, kappa and lambda chain loci, with the plan to cross the Transfer to pseudopregnant females resulting strains with each other and with transgenic rats generated with BACs containing the sequences for 5 months human Ig heavy or light chains, thus generating rats Tail biopsy and DNA analysis for ZFN activity and with fully humanized humoral immune responses. sequence The relative high efficiency of our procedure supports the possibility of generating whole animal If only one allele targeted cross- knockin rats by HR resulting from the simultaneous breed descendence to obtain homozygous deficient animals delivering into the zygote of both a nuclease and the donor-additive DNA sequence. In Drosophila, HR Fig. 2 Procedure for the generation of knockout rats. The pair using ZFNs was more efficient when using the circular of ZFNs for IgM were assembled in cis in an expression form of the donor DNA as compared to the linear one plasmid separated by a 2A self-splicing sequence (allowing separate translation from one transcript), the CAG promoter (Beumer et al. 2008); linear DNA may be more (CAGp, which is expressed in one cell embryos) and a polyA degraded by nucleases or concatenated and rendered sequence (2.87 Kb total). The ZFNs were microinjected either less capable of HR. Circular versus linear forms of the as circular plasmid (to avoid genomic insertion) or as mRNA donor DNA will have to be tested in mammalian following in vitro transcription. The plasmid DNA was microinjected into the male pronuclei and the mRNA either embryos. Also in Drosophila, deficiency of DNA ligase in the male pronuclei or in the cytoplasm. Surviving embryos IV, part of the NHEJ mechanism, increased HR were transferred to foster mothers, the DNA from newborns dramatically (Beumer et al. 2008) but DNA ligase IV tail biopsies were analyzed using the Cel-I assay to detect deficiency is lethal in mice (Barnes et al. 1998). mutations and confirmed by DNA sequencing. DNA sequenc- ing will reveal whether the animal is mutated in one or both Mutation of genes of other proteins involved in NHEJ alleles and whether the mutation is unique or multiple. and compatible with embryo survival may be a major Heterozygous mutated animals are breed with wild-type future objective to facilitate the use of ZFNs or other animals, the offspring is analyzed to confirm the transmission genes to direct HR in mammal zygotes. of the mutation (at this stage only one mutation) and to cross- breed to obtain an homozygous knockout. The whole process Along this line, nucleases could be used to direct can be done in 5 months HR to target integration of a new transgene into a 123 Transgenic Res (2010) 19:363–371 367

Table 1 Transgenic Construct Route Dose Transferred embryos Newborns % Mutants % efficiency of microinjection (ng/ml) % (total injected) (total transferred) (total borns) of ZFNs for rat IgM Plasmid PNI 10 80.9 (609) 11 (493) 11.1 (54) Plasmid PNI 2 77.3 (605) 17.5 (468) 9.8 (82) Plasmid PNI 0.4 82.8(511) 14.7 (423) 6.5 (62) mRNA PNI 10 55.9 (186) 13.5 (104) 28.6 (14) mRNA PNI 2 60.5 (832) 19.1(503) 11.5 (96) Plasmid circular plasmid to mRNA PNI 0.4 64.5 (183) 16.1 (118) 5.3 (19) avoid genomic insertion, mRNA Cytoplasmic 10 72 (272) 2 (197) 75 (4) mRNA in vitro translated, mRNA Cytoplasmic 2 68 (197) 12.7 (134) 11.7 (17) PNI pronuclear injection

Table 2 IgM mutant germ line transmission are known to be promiscuous due to the nature of the Founder No. of No. of mutants FokI endonuclease cleavage domain. The FokI offspring (% of offspring) endonuclease cleavage domain must dimerize to create an active endonuclease complex able to 0019 7 5 (71) generate a double strand break, and in its native 0046 6 1 (17) form is capable of doing so as homodimers even 0008 5 0 (0) when not bound to DNA (Mani et al. 2005), albeit 0006 6 3 (50) weakly. Even when newer generation of heterodimer- 0119 0 0 forced FokI domains are utilized (Geurts et al. 2009b; 4.1 12 1 Szczepek et al. 2007), expression of these nucleases 9.2 40 17 in a cell may result in not only the intended, but also other double strand breaks and consequent mutations. permissive locus that possesses ubiquitous or tissue- In this regard, the manifestation of toxicities related restricted expression, as has been accomplished via to ZFN injection into zebrafish embryos (Foley et al. HR in ES cells (Bronson et al. 1996). In this way, any 2009) provides confirmatory evidence that ZFN can transgene integrated by HR in such a locus would induce double strand breaks even at non-canonical reproduce its expression and site-dependent epige- sites. This is the reason why present strategies, netic modulation of transgene expression would be including our application of ZFN’s in rats, favor avoided. short-term expression of ZFNs rather than longer Other technical possibilities include the use of term expression that would provide additional time pairs of nucleases directed to two different sequences for off target cleavage to lead to additional mutations of a gene separated by large stretches of DNA in and toxic effects. Investigators interested in utilizing order to generate long deletions. ZFN-mediated gene targeting must thus consider how best to manage mutations generated in sequences other than the ones targeted: either, specific tech- Potential barriers for the application niques may be applied to detect them (Geurts et al. of nuclease-induced genetic modifications 2009b; Perez et al. 2008), or crossbreeding may be pursued to eliminate them. To what degree off target While application of nuclease-mediated gene target- cleavage and consequent mis-targeting of mutations ing in organisms ranging from plants, to insects, to will apply to engineered homing endonucleases is not Xenopus to zebrafish and now mammals has now presently known. been shown to be possible, a major open question is DNA double strand breaks are recognized by a how widely nuclease technologies can be success- complex machinery that creates epigenetic modifica- fully applied in more complex organisms. tions extending for many kilobases in either direction Zinc-finger nucleases (ZFNs), while intended to from the DNA double strand break, the recruitment make a double strand break at a specific target site, and assembly of an array of DNA repair proteins into 123 368 Transgenic Res (2010) 19:363–371 a repair complex, and signaling to cell cycle regula- genes and non-coding RNAs. However, a mammalian tory mechanisms to slow or halt cell cycle progres- embryo must not only manage its own development, sion so as to prevent unrepaired damage from being it must do so in conjunction with a placenta which is propagated to future generations of cells (Batista in turn interacting with the maternal body and et al. 2009; Donigan and Sweasy 2009; Pandita and immune system. Mammalian embryonic develop- Richardson 2009). The extent of epigenetic modifi- ment is thus significantly more complex than other cations and cell cycle alterations accompanying vertebrates, as it depends on precise quantitative induction of a DNA double strand break and their regulation, timing and coordinated expression of each influence on cell physiology may also vary according involved transcriptional unit for both appropriate to the gene expression pattern in a given cell. In the somatic development as well as the coordination of context of a germ cell/oocyte/embryo a DNA double interactions between the placenta and the maternal strand break could result in substantial chromosomal environment (Phillips and McKinnon 2007). This instability and thus prevent further developmental complexity represents another significant barrier to progression (e.g., Lo et al. 2002). extrapolation of results from insects, Xenopus,or NHEJ is the dominant DNA repair mechanism in zebrafish to mammalian embryos, as the DNA somatic cells and HR tends to be upregulated in damage response induced by even a single DNA mouse ES cells (Derijck et al. 2008). Whether NHEJ- double strand break would have multiple additional or HR-mediated resolution of a nuclease-induced opportunities to disrupt development. break is favored in early embryos and how NHEJ- or A final issue for investigators contemplating the HR can be manipulated are important areas for future use of nuclease-mediated gene targeting is that investigation. success at any single locus may not predict success Differences in response to DNA damage between at other loci—variability in gene targeting success at organisms may also hinder application of nuclease- different loci is a well known phenomenon in murine mediated gene targeting at the embryo or other stages ES cells. Thus, success at any given loci in any given of development. Data from induction of double strand organism, does not translate to an obvious potential breaks in gonocytes and spermatogonial precursors for success in gene targeting in the same organism in via gamma irradiation suggests that such cells are another system—i.e., success in targeting the zebra- highly sensitive to induction of apoptosis via double fish Ig locus could not be construed as predictive at strand breaks (e.g., Forand et al. 2009), representing a success in targeting the Ig locus in the rat, and success mechanism through which an organism prevents in the rat may likewise not be extrapolated to other genetic changes resulting from double strand breaks mammals (Danilova et al. 2005). Even our success at from being transmitted to its offspring. An additional multiple loci in rats does not guarantee success at instructive example is that zebrafish have been shown other loci. A major issue in this regard is that the to have a higher overall DNA repair capacity than capacity of a nuclease to hit an intended target site mice or humans (Sussman 2007). Thus, the effect of a may be subject to the epigenetic status of the locus double strand break during zebrafish embryonic containing the target site—e.g., in the most simplistic development would be expected to be less disruptive case, is a target in euchromatin or heterochromatin? versus mouse or rat embryonic development, which Thus, even if a given target is accessible to a targeted along with the reduced complexity of zebrafish nuclease in the context of one cell type, one could not embryonic development, renders results with nucle- know that the same target would be accessible with ase-mediated targeting difficult to extrapolate from comparable efficiency, or even at all, to the same one species to another. nuclease in another context. This would be a partic- For application to a given organism, the complex- ular concern for extrapolating data from cultured cell ity of the embryonic developmental process is also models to primary cells of any type, as cultured cell likely to be an important influence on success. Like models are often tumor derived, and are notorious for other vertebrates, the developmental program through accumulating genetic and epigenetic changes over which a mammalian embryo develops into a viable time in culture. In addition, due to an unknown fetus is dependent on complex genetic and epigenetic efficiency of cleavage, the rapid proliferation rate of processes involving hundreds, if not thousands, of early development and cell cycle alterations caused by 123 Transgenic Res (2010) 19:363–371 369 double strand break induction, the likelihood of Barnes DE, Stamp G, Rosewell I, Denzel A, Lindahl T (1998) cleavage of a specific locus in early development is Targeted disruption of the gene encoding DNA ligase IV leads to lethality in embryonic mice. Curr Biol 8(25): unknowable prior to performing the experiment in the 1395–1398 specific embryos/oocytes/germ cell system of interest. Batista LF, Kaina B, Meneghini R, Menck CF (2009) How DNA lesions are turned into powerful killing structures: insights from UV-induced apoptosis. Mutat Res 681(2–3):197–208 Beerli RR, Barbas CF III (2002) Engineering polydactyl zinc- Conclusions finger transcription factors. Nat Biotechnol 20(2):135–141 Beumer KJ, Trautman JK, Bozas A, Liu J-L, Rutter J, Gall JG, In conclusion, ZFNs, homing endonucleases, and Carroll D (2008) Efficient gene targeting in Drosophila by related nuclease technologies represent powerful and direct embryo injection with zinc-finger nucleases. Proc Natl Acad Sci USA 105(50):19821–19826 versatile new tools in the genetic engineering toolkit, Bibikova M, Golic M, Golic KG, Carroll D (2002) Targeted including gene therapy (Carroll 2008). If the objec- chromosomal cleavage and mutagenesis in Drosophila using tive is to generate targeted-gene knockouts, ZFNs in zinc-finger nucleases. Genetics 161(3):1169–1175 particular but possibly also other nucleases-mediated Bibikova M, Beumer K, Trautman JK, Carroll D (2003) Enhancing gene targeting with designed zinc finger nuc- targeted gene modification are not only crucial in leases. Science 300(5620):764 species where ES cells or cloning by NT are not Bozas A, Beumer KJ, Trautman JK, Carroll D (2009) Genetic available, but are a real alternative to these other analysis of zinc-finger nuclease-induced gene targeting in technologies in terms of speed, cost and labor in Drosophila. Genetics 182(3):641–651 Bronson SK, Plaehn EG, Kluckman KD, Hagaman JR, Maeda species in which they are available. If the objective is N, Smithies O (1996) Single-copy transgenic mice with to generate gene-additions by HR, nuclease-mediated chosen-site integration. Proc Natl Acad Sci USA 93(17): gene targeting has already been shown in non- 9067–9072 mammal species to be indispensable and has the Buehr M, Meek S, Blair K, Yang J, Ure J, Silva J, McLay R, Hall J, Ying Q, Smith A (2008) Capture of authentic embryonic potential for widespread application for this purpose stem cells from rat blastocysts. Cell 135(7):1287–1298 in mammals as an alternative to ES cells or cloning Cai C, Doyon Y, Ainley W, Miller J, DeKelver R, Moehle E, by NT. Rock J, Lee Y-L, Garrison R, Schulenberg L, Blue R, Worden A, Baker L, Faraji F, Zhang L, Holmes M, Rebar Acknowledgments This work was in part funded by the E, Collingwood T, Rubin-Wilson B, Gregory P, Urnov F, Re´gion Pays de la Loire through Biogenouest and IMBIO Petolino J (2009) Targeted transgene integration in plant programs as well as by the IBiSA program, and NIH grant cells using designed zinc finger nucleases. Plant Mol Biol UL1DE019582 to AMS. 69(6):699 Cannata F, Brunet E, Perrouault L, Roig V, Ait-Si-Ali S, Asseline U, Concordet JP, Giovannangeli C (2008) Tri- plex-forming oligonucleotide-orthophenanthroline conju- gates for efficient targeted genome modification. Proc References Natl Acad Sci USA 105(28):9576–9581 Carroll D (2008) Progress and prospects: zinc-finger nucleases Aitman TJ, Critser JK, Cuppen E, Dominiczak A, Fernandez- as gene therapy agents. Gene Ther 15(22):1463–1468 Suarez XM, Flint J, Gauguier D, Geurts AM, Gould M, Carroll D, Beumer KJ, Morton JJ, Bozas A, Trautman JK Harris PC, Holmdahl R, Hubner N, Izsvak Z, Jacob HJ, (2008) Gene targeting in Drosophila and Caenorhabditis Kuramoto T, Kwitek AE, Marrone A, Mashimo T, Mo- elegans with zinc-finger nucleases. Methods Mol Biol reno C, Mullins J, Mullins L, Olsson T, Pravenec M, Riley 435:63–77 L, Saar K, Serikawa T, Shull JD, Szpirer C, Twigger SN, Charreau B, Tesson L, Soulillou JP, Pourcel C, Anegon I Voigt B, Worley K (2008) Progress and prospects in rat (1996) Transgenic rats: technical aspects and models. genetics: a community view. Nat Genet 40(5):516–522 Transgenic Res 5:223–234 Arnould S, Chames P, Perez C, Lacroix E, Duclert A, Epinat Cozzi J, Wan E, Jacquet C, Fraichard A, Cherifi Y, Zhou Q (2009) JC, Stricher F, Petit AS, Patin A, Guillier S, Rolland S, Procedures for somatic cell nuclear transfer in the rat. Methods Prieto J, Blanco FJ, Bravo J, Montoya G, Serrano L, in mol biol ‘‘rat genomics: gene identification, functional Duchateau P, Paques F (2006) Engineering of large genomics and model applications’’, vol 561, pp 73–88 numbers of highly specific homing endonucleases that Danilova N, Bussmann J, Jekosch K, Steiner LA (2005) The induce recombination on novel DNA targets. J Mol Biol immunoglobulin heavy-chain locus in zebrafish: identifi- 355(3):443–458 cation and expression of a previously unknown isotype, Ashworth J, Havranek JJ, Duarte CM, Sussman D, Monnat RJ immunoglobulin Z. Nat Immunol 6(3):295–302 Jr, Stoddard BL, Baker D (2006) Computational redesign Derijck A, van der Heijden G, Giele M, Philippens M, de Boer of endonuclease DNA binding and cleavage specificity. P (2008) DNA double-strand break repair in parental Nature 441(7093):656–659 chromatin of mouse zygotes, the first cell cycle as an 123 370 Transgenic Res (2010) 19:363–371

origin of de novo mutation. Hum Mol Genet 17(13):1922– Kim YG, Shi Y, Berg JM, Chandrasegaran S (1997) Site-specific 1937 cleavage of DNA-RNA hybrids by zinc finger/FokI cleav- Donigan KA, Sweasy JB (2009) Sequence context-specific age domain fusions. Gene 203(1):43–49 mutagenesis and base excision repair. Mol Carcinog Kitada K, Ishishita S, Tosaka K, Takahashi R, Ueda M, Keng 48(4):362–368 VW, Horie K, Takeda J (2007) Transposon-tagged Doyon Y, McCammon JM, Miller JC, Faraji F, Ngo C, Katibah mutagenesis in the rat. Nat Methods 4(2):131–133 GE, Amora R, Hocking TD, Zhang L, Rebar EJ, Gregory Kitamura D, Roes J, Kuhn R, Rajewsky K (1991) A B cell- PD, Urnov FD, Amacher SL (2008) Heritable targeted deficient mouse by targeted disruption of the membrane gene disruption in zebrafish using designed zinc-finger exon of the immunoglobulin mu chain gene. Nature 350 nucleases. Nat Biotechnol 26(6):702 (6317):423–426 Foley JE, Yeh J-RJ, Maeder ML, Reyon D, Sander JD, Pet- Li P, Tong C, Mehrian-Shai R, Jia L, Wu N, Yan Y, Maxson erson RT, Joung JK (2009) Rapid mutation of endogenous RE, Schulze EN, Song H, Hsieh CL, Pera MF, Ying Q zebrafish genes using zinc finger nucleases made by oli- (2008) Germline competent embryonic stem cells derived gomerized pool engineering (OPEN). PLoS ONE 4(2): from rat blastocysts. Cell 135:1299–1310 e4348 Lo AW, Sprung CN, Fouladi B, Pedram M, Sabatier L, Ricoul M, Forand A, Fouchet P, Lahaye JB, Chicheportiche A, Habert R, Reynolds GE, Murnane JP (2002) instability Bernardino-Sgherri J (2009) Similarities and differences as a result of double-strand breaks near telomeres in mouse in the in vivo response of mouse neonatal gonocytes and embryonic stem cells. Mol Cell Biol 22(13):4836–4850 spermatogonia to genotoxic stress. Biol Reprod 80(5): Lois C, Hong EJ, Pease S, Brown EJ, Baltimore D (2002) 860–873 Germline transmission and tissue-specific expression of Geurts AM, Cost C, Re´my S, Cui X, Tesson L, Usal C, transgenes delivered by lentiviral vectors. Science 295 Menoret S, Jacob H, Anegon I, Buelow R (2009a) Gen- (5556):868–872 eration of gene-specific mutated rats using zinc finger Lombardo A, Genovese P, Beausejour CM, Colleoni S, Lee YL, nucleases. Methods in mol biol rat genomics: gene iden- Kim KA, Ando D, Urnov FD, Galli C, Gregory PD, Holmes tification, functional genomics and model applications. MC, Naldini L (2007) Gene editing in human stem cells Humana Press (in press) using zinc finger nucleases and integrase-defective lentiv- Geurts AM, Cost GJ, Miller JC, Freyvert Y, Zeitler B, Choi iral vector delivery. Nat Biotechnol 25(11):1298–1306 VM, Jenkins SS, Wood A, Cui X, Meng X, Vincent A, Maeder ML, Thibodeau-Beganny S, Osiak A, Wright DA, Lam S, DeKelver RC, Michalkiewicz M, Schilling R, Anthony RM, Eichtinger M, Jiang T, Foley JE, Winfrey Foeckler J, Kalloway S, Weiler H, Me´noret S, Anegon I, RJ, Townsend JA, Unger-Wallace E, Sander JD, Muller- Davis GD, Sullivan P, Zhang L, Rebar EJ, Gregory PD, Lerch F, Fu F, Pearlberg J, Gobel C, Dassie JP, Pruett- Urnov FD, Jacob HJ, Buelow R (2009b) Knockout rats Miller SM, Porteus MH, Sgroi DC, Iafrate AJ, Dobbs D, produced via embryo pronuclear microinjection of McCray PB Jr, Cathomen T, Voytas DF, Joung JK (2008) designed zinc finger nucleases. Science 325(5939):433 Rapid ‘‘open-source’’ engineering of customized zinc- Grizot S, Smith J, Daboussi F, Prieto J, Redondo P, Merino N, finger nucleases for highly efficient gene modification. Villate M, Thomas S, Lemaire L, Montoya G, Blanco FJ, Mol Cell 31(2):294–301 Paques F, Duchateau P (2009) Efficient targeting of a Mani M, Smith J, Kandavelou K, Berg JM, Chandrasegaran S SCID gene by an engineered single-chain homing endo- (2005) Binding of two zinc finger nuclease monomers to two nuclease. Nucleic Acids Res. doi:10.1093/nar/gkp548 specific sites is required for effective double-strand DNA Hockemeyer D, Soldner F, Beard C, Gao Q, Mitalipova M, cleavage. Biochem Biophys Res Commun 334(4): 1191–1197 DeKelver RC, Katibah GE, Amora R, Boydston EA, Zeitler Mattei E, Corbi N, Di Certo MG, Strimpakos G, Severini C, B, Meng X, Miller JC, Zhang L, Rebar EJ, Gregory PD, Onori A, Desantis A, Libri V, Buontempo S, Floridi A, Urnov FD, Jaenisch R (2009) Efficient targeting of Fanciulli M, Baban D, Davies KE, Passananti C (2007) expressed and silent genes in human ESCs and iPSCs using Utrophin up-regulation by an artificial transcription factor zinc-finger nucleases. Nat Biotechnol 27(9):851–857 in transgenic mice. PLoS ONE 2(1):e774 Isalan M, Klug A, Choo Y (2001) A rapid, generally applicable Me´noret S, Remy S, Usal C, Tesson L, Anegon I (2009) method to engineer zinc fingers illustrated by targeting the Generation of transgenic rats by microinjection of short HIV-1 promoter. Nat Biotechnol 19(7):656–660 DNA fragments. Methods in mol biol ‘‘rat genomics: gene Jacob HJ (2009) The rat: a model used in biomedical research. identification, functional genomics and model applica- Methods in mol biol ‘‘rat genomics: gene identification, tions’’. Humana Press (in press) functional genomics and model applications’’. Humana Miller JC, Holmes MC, Wang J, Guschin DY, Lee YL, Rup- Press (in press) niewski I, Beausejour CM, Waite AJ, Wang NS, Kim KA, Kandavelou K, Chandrasegaran S (2009) Custom-designed Gregory PD, Pabo CO, Rebar EJ (2007) An improved molecular scissors for site-specific manipulation of the zinc-finger nuclease architecture for highly specific gen- plant and Mammalian . Methods Mol Biol 544: ome editing. Nat Biotechnol 25(7):778–785 617–636 Moehle EA, Rock JM, Lee YL, Jouvenot Y, DeKelver RC, Kawamata M, Ochiya T (2009) Establishment of embryonic Gregory PD, Urnov FD, Holmes MC (2007) Targeted stem cells from rat blastocysts. Methods in mol biol ‘‘rat gene addition into a specified location in the human genomics: gene identification, functional genomics and genome using designed zinc finger nucleases. Proc Natl model applications’’. Humana Press (in press) Acad Sci USA 104(9):3055–3060

123 Transgenic Res (2010) 19:363–371 371

Monnat R Jr, Scharenberg A, Stoddard B (2008) Progress in Stoddard BL, Scharenberg AM, Monnat RJ Jr (2008) Advances engineering homing endonucleases for gene targeting: ten in engineering homing endonucleases for gene targeting: years after structures. In: Bertolotti R, Ozawa K (eds) Pro- ten years after structures. In: Bertolotti R, Ozawa K (eds) gress in gene therapy volume 3: autologous and cancer stem Chapter 6 in progress in gene therapy 3: autologous and cell gene therapy. World Scientific Publishers, Singapore cancer stem cell gene therapy. World Scientific Press, Mullins JJ, Peters J, Ganten D (1990) Fulminant hypertension Hackensack, NJ, pp 135–167 in transgenic rats harbouring the mouse Ren-2 gene. Sussman R (2007) DNA repair capacity of zebrafish. Proc Natl Nature 344:541–544 Acad Sci USA 104(33):13379–13383 Pabo CO, Peisach E, Grant RA (2001) Design and selection of Szczepek M, Brondani V, Buchel J, Serrano L, Segal DJ, Cath- novel Cys2His2 zinc finger proteins. Annu Rev Biochem omen T (2007) Structure-based redesign of the dimerization 70(1):313–340 interface reduces the toxicity of zinc-finger nucleases. Nat Pandita TK, Richardson C (2009) Chromatin remodeling finds Biotechnol 25(7):786–793 its place in the DNA double-strand break response. Takeuchi R, Certo M, Caprara MG, Scharenberg AM, Stoddard Nucleic Acids Res 37(5):1363–1377 BL (2009) Optimization of in vivo activity of a bifunctional Paques F, Duchateau P (2007) Meganucleases and DNA dou- homing endonuclease and maturase reverses evolutionary ble-strand break-induced recombination: perspectives for degradation. Nucleic Acids Res 37(3):877–890 gene therapy. Curr Gene Ther 7(1):49–66 Tesson L, Cozzi J, Menoret S, Remy S, Usal C, Fraichard A, Perez EE, Wang J, Miller JC, Jouvenot Y, Kim KA, Liu O, Anegon I (2005) Transgenic modifications of the rat Wang N, Lee G, Bartsevich VV, Lee Y-L, Guschin DY, genome. Transgenic Res 14(5):531–546 Rupniewski I, Waite AJ, Carpenito C, Carroll RG, Orange Townsend JA, Wright DA, Winfrey RJ, Fu F, Maeder ML, JS, Urnov FD, Rebar EJ, Ando D, Gregory PD, Riley JL, Joung JK, Voytas DF (2009) High-frequency modification Holmes MC, June CH (2008) Establishment of HIV-1 of plant genes using engineered zinc-finger nucleases. resistance in CD4? T cells by genome editing using zinc- Nature 459(7245):442 finger nucleases. Nat Biotechnol 26(7):808 Ueda S, Kawamata M, Teratani T, Shimizu T, Tamai Y, Ogawa Pfeifer A (2006) Lentiviral transgenesis—a versatile tool for H, Hayashi K, Tsuda H, Ochiya T (2008) Establishment of basic research and gene therapy. Curr Gene Ther 6(4): rat embryonic stem cells and making of chimera rats. PLoS 535–542 One 3(7):e2800 Phillips ER, McKinnon PJ (2007) DNA double-strand break Urnov FD, Miller JC, Lee Y-L, Beausejour CM, Rock JM, repair and development. Oncogene 26(56):7799–7808 Augustus S, Jamieson AC, Porteus MH, Gregory PD, Porteus M (2008). Design and testing of zinc finger nucleases for Holmes MC (2005) Highly efficient endogenous human gene use in mammalian cells. Methods in Mol Biol.‘‘Chromo- correction using designed zinc-finger nucleases. Nature 435 somal mutagenesis’’, vol 435. Humana Press, pp 47–61 (7042):646 Re´my S, NGuyen T, Me´noret S, Tesson L, Usal C, Anegon I Volna P, Jarjour J, Baxter S, Roffler SR, Monnat RJ Jr, Stoddard (2009) The use of lentiviral vectors to obtain transgenic BL, Scharenberg AM (2007) Flow cytometric analysis of rats. Methods in mol biol rat genomics: gene identifica- DNA binding and cleavage by cell surface-displayed tion, functional genomics and model applications. Hu- homing endonucleases. Nucleic Acids Res 35(8):2748– mana Press (in press) 2758 Sander JD, Zaback P, Joung JK, Voytas DF, Dobbs D (2007) Wright DA, Thibodeau-Beganny S, Sander JD, Winfrey RJ, Zinc Finger Targeter (ZiFiT): an engineered zinc finger/ Hirsh AS, Eichtinger M, Fu F, Porteus MH, Dobbs D, target site design tool. Nucleic Acids Res 35((suppl_2)): Voytas DF, Joung JK (2006) Standardized reagents and W599–W605 protocols for engineering zinc finger nucleases by mod- Santiago Y, Chan E, Liu PQ, Orlando S, Zhang L, Urnov FD, ular assembly. Nat Protocols 1(4):1637 Holmes MC, Guschin D, Waite A, Miller JC, Rebar EJ, Yang M, Djukanovic V, Stagg J, Lenderts B, Bidney D, Falco Gregory PD, Klug A, Collingwood TN (2008) Targeted SC, Lyznik LA (2009) Targeted mutagenesis in the gene knockout in mammalian cells by using engineered progeny of transgenic plants. Plant Mol Biol 70(6): zinc-finger nucleases. Proc Natl Acad Sci USA 105(15): 669–679 5809–5814 Zan Y, Haag JD, Chen KS, Shepel LA, Wigington D, Wang YR, Shinohara T, Kato M, Takehashi M, Lee J, Chuma S, Nakatsuji Hu R, Lopez-Guajardo CC, Brose HL, Porter KI, Leonard N, Kanatsu-Shinohara M, Hirabayashi M (2006) Rats RA, Hitt AA, Schommer SL, Elegbede AF, Gould MN produced by interspecies spermatogonial transplantation (2003) Production of knockout rats using ENU mutagenesis in mice and in vitro microinsemination. Proc Natl Acad and a yeast-based screening assay. Nat Biotechnol 21(6): Sci USA 103(37):13624–13628 645–651 Shukla VK, Doyon Y, Miller JC, DeKelver RC, Moehle EA, Zhou Q, Renard JP, Le Friec G, Brochard V, Beaujean N, Worden SE, Mitchell JC, Arnold NL, Gopalan S, Meng X, Cherifi Y, Fraichard A, Cozzi J (2003) Generation of Choi VM, Rock JM, Wu Y-Y, Katibah GE, Zhifang G, fertile cloned rats by regulating oocyte activation. Science McCaskill D, Simpson MA, Blakeslee B, Greenwalt SA, 302:1179 Butler HJ, Hinkley SJ, Zhang L, Rebar EJ, Gregory PD, Zou X, Ayling C, Xian J, Piper TA, Barker PJ, Bruggemann M Urnov FD (2009) Precise genome modification in the crop (2001) Truncation of the mu heavy chain alters BCR sig- species Zea mays using zinc-finger nucleases. Nature nalling and allows recruitment of CD5? B cells. Int 459(7245):437 Immunol 13(12):1489–1499

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