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
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
Recently, a series of genome editing technologies including ZFNs, TALENs, and CRISPR/Cas9 systems have enabled gene modification in the endogenous target genes of various organisms including pigs, which are important for agricultural and biomedical research. Owing to its simple application for gene knockout and ease of use, the CRISPR/Cas9 is now in common use worldwide. The most important aspect of this process is the selection of the method used to deliver genome editing components to embryos. In earlier stages, zygote microinjection of these components [single guide RNA (sgRNA) + DNA/mRNA for Cas9] into the cytoplasm and/or nuclei of a zygote has been frequently employed. However, this method is always associated with the generation of mosaic embryos in which genome-edited and unedited cells are mixed together. To avoid this mosaic issue, in vitro electroporation of zygotes in the presence of sgRNA mixed with Cas9 protein, referred to as a ribonucleoprotein (RNP), is now in frequent use. This review provides a historical background of the production of genome-edited pigs and also presents current research concerning how genome editing is induced in somatic cell nuclear transfer-derived embryos that have been reconstituted with normal nuclei.
Part of the book: Reproductive Biology and Technology in Animals