Shimyureta to jikki o mochiita haiburiddo-gata kikai gakushuho ni kansuru kenkyu

制度:新 ; 報告番号:甲2816号 ; 学位の種類:博士(工学) ; 授与年月日:2009/2/25 ; 早大学位記番号:新503

Possible Production of Genome-Edited Animals Using Gene-Engineered Sperm

CRISPR/Cas9 is widely used for genome editing in a variety of organisms, including mammals, fishes, and plants. In mammals, zygotes are considered an appropriate target for gene delivery of CRISPR/Cas9 components [Cas9 endonuclease and a single-guide (sgRNA)] via microinjection or in vitro electroporation. However, these approaches require ex vivo handling of zygotes, which is necessary for egg transfer to recipient females to allow the treated zygotes to develop full-term. These procedures are often laborious, time-consuming, and use numerous mice. In our previous experiments, the plasmid DNA encapsulated by liposomal reagent introduced into the internal portion of a testis can be transferred to the mature sperm present in the epididymal ducts, and is finally transferred to oocytes via fertilization. Although it was not integrated into their genome, this approach would be useful for creating genome-edited animals, since CRISPR/Cas9 can be performed by transient interaction of Cas9 and sgRNA, whereby chromosomal integration of the CRISPR components is not a prerequisite. Here, we will review past achievements concerning in vivo transfection of immature/mature sperm and present experimental proposals for possible genome editing via gene-engineered sperm based on recent findings

Intraoviductal Instillation of a Solution as an Effective Route for Manipulating Preimplantation Mammalian Embryos in vivo

Preimplantation embryos of mammals are enclosed by a translucent layer called zona pellucida (ZP), which is composed of glycoproteins. ZP is important for protecting against infection by virus and bacteria, and to prevent attachment of embryos to the oviductal epithelia. Due to the presence of ZP, it has been difficult to transfect preimplantation embryos existing within the oviductal lumen, with exogenous nucleic acids, such as DNA and mRNA. However, intraoviductal instillation of nucleic acids, and subsequent in vivo electroporation in pregnant females, enables transfection of these embryos, leading to the production of gene-modified animals. This new method for production of genetically modified animals does not require any ex vivo handling of embryos, which has been essential for traditional transgenesis. In this article, we describe recent advances in the in vivo transfection of preimplantation mammalian embryos, and also the possibility of simple transfection of these embryos through intraoviductal instillation of a solution, alone

Enantioselective Protonation of Ketene Bis(trimethylsilyl) Acetals Derived from α-Aryl-α-haloacetic Acids Using LBA

Optically active α-halocarboxylic acids and derivatives are important and versatile building blocks in organic synthesis. Lewis acid assisted chiral Brönsted acid (LBA) was recently prepared in situ from tin(IV) tetrachloride and optically pure binaphthol and shown to be an effective reagent for enantioselective protonation of pro- chiral silyl enol ethers and ketene bis(trialkylsilyl) acetals. In this paper we describe highly enantioselective protonation of ketene bis(trimethylsilyl) acetals prepared from α-aryl-α-haloaeetic acids using LBA. This is a new methodology for the enantioselective synthesis of α-aryl-α-haloacetic acid derivatives, and the commercially available chiral binaphthol can be recovered efficiently for reuse

Anisotropic uniaxial pressure response of the Mott insulator Ca2RuO4

We have investigated the in-plane uniaxial pressure effect on the antiferromagnetic Mott insulator Ca2RuO4 from resistivity and magnetization measurements. We succeeded in inducing the ferromagnetic metallic phase at lower critical pressure than by hydrostatic pressure, indicating that the flattening distortion of the RuO6 octahedra is more easily released under in-plane uniaxial pressure. We also found a striking in-plane anisotropy in the pressure responses of various magnetic phases: Although the magnetization increases monotonically with pressure diagonal to the orthorhombic principal axes, the magnetization exhibits peculiar dependence on pressure along the in-plane orthorhombic principal axes. This peculiar dependence can be explained by a qualitative difference between the uniaxial pressure effects along the orthorhombic a and b axes, as well as by the presence of twin domain structures.Comment: Accepted for publication in Phys. Rev.