Generation of Knockout Rats with X-Linked Severe Combined Immunodeficiency (X-SCID) Using Zinc-Finger Nucleases

Background: Although the rat is extensively used as a laboratory model, the inability to utilize germ line-competent rat embryonic stem (ES) cells has been a major drawback for studies that aim to elucidate gene functions. Recently, zinc-finger nucleases (ZFNs) were successfully used to create genome-specific double-stranded breaks and thereby induce targeted gene mutations in a wide variety of organisms including plants, drosophila, zebrafish, etc. Methodology/Principal Findings: We report here on ZFN-induced gene targeting of the rat interleukin 2 receptor gamma (Il2rg) locus, where orthologous human and mouse mutations cause X-linked severe combined immune deficiency (X-SCID). Co-injection of mRNAs encoding custom-designed ZFNs into the pronucleus of fertilized oocytes yielded genetically modified offspring at rates greater than 20%, which possessed a wide variety of deletion/insertion mutations. ZFN-modified founders faithfully transmitted their genetic changes to the next generation along with the severe combined immune deficiency phenotype. Conclusions and Significance: The efficient and rapid generation of gene knockout rats shows that using ZFN technology is a new strategy for creating gene-targeted rat models of human diseases. In addition, the X-SCID rats that were established in this study will be valuable in vivo tools for evaluating drug treatment or gene therapy as well as model systems for examining the treatment of xenotransplanted malignancies

Numerical simulation of the impact of COVID-19 lockdown on tropospheric composition and aerosol radiative forcing in Europe

Aerosols influence the Earth\u27s energy balance directly by modifying the radiation transfer and indirectly by altering the cloud microphysics. Anthropogenic aerosol emissions dropped considerably when the global COVID-19 pandemic resulted in severe restraints on mobility, production, and public life in spring 2020. We assess the effects of these reduced emissions on direct and indirect aerosol radiative forcing over Europe, excluding contributions from contrails. We simulate the atmospheric composition with the ECHAM5/MESSy Atmospheric Chemistry (EMAC) model in a baseline (business-as-usual) and a reduced emission scenario. The model results are compared to aircraft observations from the BLUESKY aircraft campaign performed in May–June 2020 over Europe. The model agrees well with most of the observations, except for sulfur dioxide, particulate sulfate, and nitrate in the upper troposphere, likely due to a biased representation of stratospheric aerosol chemistry and missing information about volcanic eruptions. The comparison with a baseline scenario shows that the largest relative differences for tracers and aerosols are found in the upper troposphere, around the aircraft cruise altitude, due to the reduced aircraft emissions, while the largest absolute changes are present at the surface. We also find an increase in all-sky shortwave radiation of 0.21 ± 0.05 W m⁻² at the surface in Europe for May 2020, solely attributable to the direct aerosol effect, which is dominated by decreased aerosol scattering of sunlight, followed by reduced aerosol absorption caused by lower concentrations of inorganic and black carbon aerosols in the troposphere. A further increase in shortwave radiation from aerosol indirect effects was found to be much smaller than its variability. Impacts on ice crystal concentrations, cloud droplet number concentrations, and effective crystal radii are found to be negligible

SsODN-mediated knock-in with CRISPR-Cas for large genomic regions in zygotes

The CRISPR-Cas system is a powerful tool for generating genetically modified animals; however, targeted knock-in (KI) via homologous recombination remains difficult in zygotes. Here we show efficient gene KI in rats by combining CRISPR-Cas with single-stranded oligodeoxynucleotides (ssODNs). First, a 1-kb ssODN co-injected with guide RNA (gRNA) and Cas9 messenger RNA produce GFP-KI at the rat Thy1 locus. Then, two gRNAs with two 80-bp ssODNs direct efficient integration of a 5.5-kb CAG-GFP vector into the Rosa26 locus via ssODN-mediated end joining. This protocol also achieves KI of a 200-kb BAC containing the human SIRPA locus, concomitantly knocking out the rat Sirpa gene. Finally, three gRNAs and two ssODNs replace 58-kb of the rat Cyp2d cluster with a 6.2-kb human CYP2D6 gene. These ssODN-mediated KI protocols can be applied to any target site with any donor vector without the need to construct homology arms, thus simplifying genome engineering in living organisms

Efficient gene targeting by TAL effector nucleases coinjected with exonucleases in zygotes

TALエフェクターヌクレアーゼ(TALEN)を用いた効率的な遺伝子改変ラットの作製技術. 京都大学プレスリリース. 2013-02-13.TAL Effector Nucleases (TALENs) are versatile tools for targeted gene editing in various species. However, their efficiency is still insufficient, especially in mammalian embryos. Here, we showed that combined expression of Exonuclease 1 (Exo1) with engineered site-specific TALENs provided highly efficient disruption of the endogenous gene in rat fibroblast cells. A similar increased efficiency of up to ~30% with Exo1 was also observed in fertilized rat eggs, and in the production of knockout rats for the albino (Tyr) gene. These findings demonstrate TALENs with Exo1 is an easy and efficient method of generating gene knockouts using zygotes, which increases the range of gene targeting technologies available to various species

A set of highly informative rat simple sequence length polymorphism (SSLP) markers and genetically defined rat strains.

[Background]The National Bio Resource Project for the Rat in Japan (NBRP-Rat) is focusing on collecting, preserving and distributing various rat strains, including spontaneous mutant, transgenic, congenic, and recombinant inbred (RI) strains. To evaluate their value as models of human diseases, we are characterizing them using 109 phenotypic parameters, such as clinical measurements, internal anatomy, metabolic parameters, and behavioral tests, as part of the Rat Phenome Project. Here, we report on a set of 357 simple sequence length polymorphism (SSLP) markers and 122 rat strains, which were genotyped by the marker set. [Results]The SSLP markers were selected according to their distribution patterns throughout the whole rat genome with an average spacing of 7.59 Mb. The average number of informative markers between all possible pairs of strains was 259 (72.5% of 357 markers), showing their high degree of polymorphism. From the genetic profile of these rat inbred strains, we constructed a rat family tree to clarify their genetic background. [Conclusion]These highly informative SSLP markers as well as genetically and phenotypically defined rat strains are useful for designing experiments for quantitative trait loci (QTL) analysis and to choose strategies for developing new genetic resources. The data and resources are freely available at the NBRP-Rat web site [1]

Daphni

Narthex, Südliches Joch, westliche Schildwand, Verkündigung an Joachim (nach Lazarides, Daphni, 1977, Abb. 2