Zinc-finger nuclease-driven targeted integration into mammalian genomes using donors with limited chromosomal homology.

We previously demonstrated high-frequency, targeted DNA addition mediated by the homology-directed DNA repair pathway. This method uses a zinc-finger nuclease (ZFN) to create a site-specific double-strand break (DSB) that facilitates copying of genetic information into the chromosome from an exogenous donor molecule. Such donors typically contain two approximately 750 bp regions of chromosomal sequence required for homology-directed DNA repair. Here, we demonstrate that easily-generated linear donors with extremely short (50 bp) homology regions drive transgene integration into 5-10% of chromosomes. Moreover, we measure the overhangs produced by ZFN cleavage and find that oligonucleotide donors with single-stranded 5' overhangs complementary to those made by ZFNs are efficiently ligated in vivo to the DSB. Greater than 10% of all chromosomes directly incorporate this exogenous DNA via a process that is dependent upon and guided by complementary 5' overhangs on the donor DNA. Finally, we extend this non-homologous end-joining (NHEJ)-based technique by directly inserting donor DNA comprising recombinase sites into large deletions created by the simultaneous action of two separate ZFN pairs. Up to 50% of deletions contained a donor insertion. Targeted DNA addition via NHEJ complements our homology-directed targeted integration approaches, adding versatility to the manipulation of mammalian genomes.

The supernova remnant SN 1006 as a Galactic particle accelerator.

The origin of cosmic rays is a pivotal open issue of high-energy astrophysics. Supernova remnants are strong candidates to be the Galactic factory of cosmic rays, their blast waves being powerful particle accelerators. However, supernova remnants can power the observed flux of cosmic rays only if they transfer a significant fraction of their kinetic energy to the accelerated particles, but conclusive evidence for such efficient acceleration is still lacking. In this scenario, the shock energy channeled to cosmic rays should induce a higher post-shock density than that predicted by standard shock conditions. Here we show this effect, and probe its dependence on the orientation of the ambient magnetic field, by analyzing deep X-ray observations of the Galactic remnant of SN 1006. By comparing our results with state-of-the-art models, we conclude that SN 1006 is an efficient source of cosmic rays and obtain an observational support for the quasi-parallel acceleration mechanism.

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

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.

Laboratory unraveling of matter accretion in young stars.

Accretion dynamics in the formation of young stars is still a matter of debate because of limitations in observations and modeling. Through scaled laboratory experiments of collimated plasma accretion onto a solid in the presence of a magnetic field, we open a first window on this phenomenon by tracking, with spatial and temporal resolution, the dynamics of the system and simultaneously measuring multiband emissions. We observe in these experiments that matter, upon impact, is ejected laterally from the solid surface and then refocused by the magnetic field toward the incoming stream. This ejected matter forms a plasma shell that envelops the shocked core, reducing escaped x-ray emission. This finding demonstrates one possible structure reconciling current discrepancies between mass accretion rates derived from x-ray and optical observations, respectively.

Bright hot impacts by erupted fragments falling back on the Sun: a template for stellar accretion.

Impacts of falling fragments observed after the eruption of a filament in a solar flare on 7 June 2011 are similar to those inferred for accretion flows on young stellar objects. As imaged in the ultraviolet (UV)-extreme UV range by the Atmospheric Imaging Assembly onboard the Solar Dynamics Observatory, many impacts of dark, dense matter display uncommonly intense, compact brightenings. High-resolution hydrodynamic simulations show that such bright spots, with plasma temperatures increasing from ~10(4) to ~10(6) kelvin, occur when high-density plasma (>>10(10) particles per cubic centimeter) hits the solar surface at several hundred kilometers per second, producing high-energy emission as in stellar accretion. The high-energy emission comes from the original fragment material and is heavily absorbed by optically thick plasma, possibly explaining the lower mass accretion rates inferred from x-rays relative to UV-optical-near infrared observations of young stars.

Cys(9), Cys(104) and Cys(207) of simian virus 40 Vp1 are essential for infectious virion formation in CV-1 cells.

Structural studies have implicated Cys(9), Cys(104) and Cys(207) of simian virus 40 (SV40) Vp1 in disulfide bond formation. Recently, we have shown the three cysteines to be essential for disulfide linkage of Vp1 complexes in vitro. Here, the role of the three cysteines was explored during the course of SV40 infection. Single-, double- and triple-mutant Vp1 at Cys(9), Cys(104) and Cys(207) continued to localize to the nuclei of transfected CV-1 cells and to bind DNA, but showed a range of abilities to form plaques. Only mutants containing the Cys(9)-->Ser change showed defects in plaque formation. Single mutants at Cys(9) formed small plaques; mutants at Cys(9). Cys(104), Cys(9). Cys(207) and Cys(9). Cys(104). Cys(207) formed no plaques. All three isolated revertants contained back-mutations at the Vp1 Cys(9) codon. These results further confirm the involvement of the three Vp1 cysteines in protein-protein interactions during virus assembly. Cys(9) is critical for production of wild-type infectious virions, whereas Cys(104) and Cys(207) play secondary roles.