Chapters authored
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.
Part of the book: New Insights into Theriogenology
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.
Part of the book: Gene Editing
Recent Advances in In Vivo Genome Editing Targeting Mammalian Preimplantation Embryos
CRISPR-based genome engineering has been widely used for producing gene-modified animals such as mice and rats, to explore the function of a gene of interest and to create disease models. However, it always requires the ex vivo handling of preimplantation embryos, as exemplified by the microinjection of genome editing components into zygotes or in vitro electroporation of zygotes in the presence of genome editing components, and subsequent cultivation of the treated embryos prior to egg transfer to the recipient females. To avoid this ex vivo process, we have developed a novel method called genome-editing via oviductal nucleic acids delivery (GONAD) or improved GONAD (i-GONAD), which enables in situ genome editing of zygotes present in the oviductal lumen of a pregnant female. This technology does not require any ex vivo handling of preimplantation embryos or preparation of recipient females and vasectomized males, all of which are often laborious and time-consuming. In this chapter, recent advances in the development of GONAD/i-GONAD will be described.
Part of the book: CRISPR Technology