Rapid "open-source" engineering of customized zinc-finger nucleases for highly efficient gene modification.

Custom-made zinc-finger nucleases (ZFNs) can induce targeted genome modifications with high efficiency in cell types including Drosophila, C. elegans, plants, and humans. A bottleneck in the application of ZFN technology has been the generation of highly specific engineered zinc-finger arrays. Here we describe OPEN (Oligomerized Pool ENgineering), a rapid, publicly available strategy for constructing multifinger arrays, which we show is more effective than the previously published modular assembly method. We used OPEN to construct 37 highly active ZFN pairs which induced targeted alterations with high efficiencies (1%-50%) at 11 different target sites located within three endogenous human genes (VEGF-A, HoxB13, and CFTR), an endogenous plant gene (tobacco SuRA), and a chromosomally integrated EGFP reporter gene. In summary, OPEN provides an "open-source" method for rapidly engineering highly active zinc-finger arrays, thereby enabling broader practice, development, and application of ZFN technology for biological research and gene therapy.

Targeted genome editing in pluripotent stem cells using zinc-finger nucleases.

Zinc-finger nucleases (ZFNs) are designer nucleases capable of cleaving a prespecified target DNA within complex genomes. ZFNs consist of a non-specific endonuclease domain fused to an engineered DNA-binding domain that tethers the nuclease activity to the chosen chromosomal site. The endonuclease-induced DNA double strand break triggers a cellular DNA damage response, resulting in double strand break repair by either accurate homologous recombination (HR) or error-prone non-homologous end-joining (NHEJ). Thus, ZFNs are powerful tools for targeted genome engineering in a variety of mammalian cell types, including embryonic (ESCs) and induced pluripotent stem cells (iPSCs). As a paradigm for genome editing in pluripotent stem cells, we describe the use of ZFNs in murine ESCs for generating knockout alleles by NHEJ without selection or by HR employing different selection schemes.

ISG15, an interferon-stimulated ubiquitin-like protein, is not essential for STAT1 signaling and responses against vesicular stomatitis and lymphocytic choriomeningitis virus.

ISG15 is an interferon-induced ubiquitin-like modifier which can be conjugated to distinct, but largely unknown, proteins. ISG15 has been implicated in a variety of biological activities, which encompass antiviral defense, immune responses, and pregnancy. Mice lacking UBP43 (USP18), the ISG15-deconjugating enzyme, develop a severe phenotype with brain injuries and lethal hypersensitivity to poly(I:C). It has been reported that an augmented conjugation of ISG15 in the absence of UBP43 induces prolonged STAT1 phosphorylation and that the ISG15 conjugation plays an important role in the regulation of JAK/STAT and interferon signaling (O. A. Malakhova, M. Yan, M. P. Malakhov, Y. Yuan, K. J. Ritchie, K. I. Kim, L. F. Peterson, K. Shuai, and D. E. Zhang, Genes Dev. 17:455-460, 2003). Here, we report that ISG15(-/-) mice are viable and fertile and display no obvious abnormalities. Lack of ISG15 did not affect the development and composition of the main cellular compartments of the immune system. The interferon-induced antiviral state and immune responses directed against vesicular stomatitis virus and lymphocytic choriomeningitis virus were not significantly altered in the absence of ISG15. Furthermore, interferon- or endotoxin-induced STAT1 tyrosine-phosphorylation, as well as expression of typical STAT1 target genes, remained unaffected by the lack of ISG15. Thus, ISG15 is dispensable for STAT1 and interferon signaling.

Selection-independent generation of gene knockout mouse embryonic stem cells using zinc-finger nucleases.

Gene knockout in murine embryonic stem cells (ESCs) has been an invaluable tool to study gene function in vitro or to generate animal models with altered phenotypes. Gene targeting using standard techniques, however, is rather inefficient and typically does not exceed frequencies of 10(-6). In consequence, the usage of complex positive/negative selection strategies to isolate targeted clones has been necessary. Here, we present a rapid single-step approach to generate a gene knockout in mouse ESCs using engineered zinc-finger nucleases (ZFNs). Upon transient expression of ZFNs, the target gene is cleaved by the designer nucleases and then repaired by non-homologous end-joining, an error-prone DNA repair process that introduces insertions/deletions at the break site and therefore leads to functional null mutations. To explore and quantify the potential of ZFNs to generate a gene knockout in pluripotent stem cells, we generated a mouse ESC line containing an X-chromosomally integrated EGFP marker gene. Applying optimized conditions, the EGFP locus was disrupted in up to 8% of ESCs after transfection of the ZFN expression vectors, thus obviating the need of selection markers to identify targeted cells, which may impede or complicate downstream applications. Both activity and ZFN-associated cytotoxicity was dependent on vector dose and the architecture of the nuclease domain. Importantly, teratoma formation assays of selected ESC clones confirmed that ZFN-treated ESCs maintained pluripotency. In conclusion, the described ZFN-based approach represents a fast strategy for generating gene knockouts in ESCs in a selection-independent fashion that should be easily transferrable to other pluripotent stem cells.

IFN-stimulated gene 15 functions as a critical antiviral molecule against influenza, herpes, and Sindbis viruses.

Type I interferons (IFNs) play an essential role in the host response to viral infection through the induction of numerous IFN-stimulated genes (ISGs), including important antiviral molecules such as PKR, RNase L, Mx, and iNOS. Yet, additional antiviral ISGs likely exist. IFN-stimulated gene 15 (ISG15) is a ubiquitin homolog that is rapidly up-regulated after viral infection, and it conjugates to a wide array of host proteins. Although it has been hypothesized that ISG15 functions as an antiviral molecule, the initial evaluation of ISG15-deficient mice revealed no defects in their responses to vesicular stomatitis virus or lymphocytic choriomeningitis virus, leaving open the important question of whether ISG15 is an antiviral molecule in vivo. Here we demonstrate that ISG15 is critical for the host response to viral infection. ISG15-/- mice are more susceptible to influenza A/WSN/33 and influenza B/Lee/40 virus infections. ISG15-/- mice also exhibited increased susceptibility to both herpes simplex virus type 1 and murine gammaherpesvirus 68 infection and to Sindbis virus infection. The increased susceptibility of ISG15-/- mice to Sindbis virus infection was rescued by expressing wild-type ISG15, but not a mutant form of ISG15 that cannot form conjugates, from the Sindbis virus genome. The demonstration of ISG15 as a novel antiviral molecule with activity against both RNA and DNA viruses provides a target for the development of therapies against important human pathogens.

Subconfluent endothelial cells form podosomes downstream of cytokine and RhoGTPase signaling.

Adhesion, migration and invasion of endothelial cells are prerequisites for the formation of blood vessels and have to be controlled on a subcellular level. We report that subconfluent human umbilical vein endothelial cells (HUVEC) are able to constitutively form podosomal adhesions that are sites of matrix metalloprotease concentration and matrix degradation. Importantly, incubation of serum-starved cells with VEGF or TNFalpha revealed the dependence of podosomes on cytokine signaling. Podosome formation was also stimulated by addition of monocytes to HUVEC. Microinjection/application of specific inhibitors or active/inactive mutants showed that regulatory pathways include Src kinase and RhoGTPase signaling, N-WASP activation and Arp2/3 complex-dependent actin nucleation. In sum, our data show that HUVEC displaying a migratory phenotype constitutively form f-actin-rich adhesions with podosomal characteristics downstream of cytokine signaling. We propose that HUVEC podosomes play an important role in endothelial cell migration and invasion.

Novel B cell population producing functional IgG in the absence of membrane IgM expression.

Surface expression of IgM is a characteristic feature of the development of most B cells. Only pre-B cells bearing functional IgM heavy chains mu chains) are selected for clonal expansion and differentiation. Cells lacking mu chains are normally eliminated. muMT mice carrying a deletion of the first exon coding for the transmembrane domain of the immunoglobulin mu chain gene were described as mice deficient for mature B cells, plasma cells and immunoglobulins in serum. In this study, we describe in muMT/BALB/c mice the presence of a novel B cell population, producing IgG, IgA and IgE in the absence of IgM membrane expression. Moreover, this small population of B cells is able to recognize antigens and to differentiate into plasma cells. These "non-conventional" mu(- / -) B cells produce functional immunoglobulins after immunization, undergo germinal center reactions, and maintain B cell memory. Our findings support the concept, that a small percentage of mu -non-expressing pre-B cells can escape elimination, switch to downstream immunoglobulin heavy chains and respond to antigens. It remains an open question how the reactivity of these B cells is regulated and in which extent such B cells play a role in physiological and pathological processes such as autoantibody production and autoimmunity.