GONAD: Genome-editing via Oviductal Nucleic Acids Delivery system: a novel microinjection independent genome engineering method in mice

Abstract

Microinjection is considered the gold standard technique for delivery of nucleic acids (NAs; transgenes or genome editing tools such as CRISPR/Cas9 systems) into embryos, for creating genetically modified organisms. It requires sophisticated equipment as wel as well-trained and highly skilled personnel to perform the micro-injection technique. Here, we describe a novel and simple microinjection-independent technique, called Genome-editing via Oviductal Nucleic Acids Delivery (GONAD). Using GONAD, we show that NAs (e.g., eGFP mRNA or Cas9 mRNA/sgRNAs) can be effectively delivered to pre-implantation embryos within the intact mouse oviduct by a simple electroporation method, and result in the desired genetic modification in the embryos. Thus GONAD can bypass many complex steps in transgenic technology such as isolation of zygotes, microinjection of NAs into them, and their subsequent transfer to pseudo-pregnant animals. Furthermore, this method can potentially be used for genome editing in species other than mice.

Leapfrogging: primordial germ cell transplantation permits recovery of CRISPR/Cas9-induced mutations in essential genes

“…Several potential solutions to this problem have been published. In rodents, the introduction of programmable nucleases, performed either in vivo or in vitro, into adult spermatogonial or oogonial stem cells has been accomplished (Fanslow et al, 2014;Chapman et al, 2015;Sato et al, 2015;Takahashi et al, 2015;Wu et al, 2015). These mutated germ cell precursors variably contribute to the germline while endogenous unmutated germ cells remain.…”

“…Leapfrogging might also be viable in birds. PGCs have been transplanted from cultured early chick extraembryonic tissue and can contribute to the germline in recipients (van de Lavoir et al, 2006;Nakamura et al, 2013). We envision combining this technology with programmable nucleases (Park et al, 2014;Véron et al, 2015) to make leapfrogging possible in this and a number of other species.…”

CRISPR/Cas9: a breakthrough in generating mouse models for endocrinologists

“…A second round of confirmatory experiments, performed on a gene-by-gene approach, could confirm the gene identification process. The rate-limiting step for such a CRISPR-based genetic screening in mice is oocyte microinjection, which could be replaced by oocyte electroporation (Hashimoto & Takemoto 2015, Qin et al 2015, Takahashi et al 2015.…”

Status Quo and Prospect of Colorectal Animal Models: Application of Transgenic Technology

“…Currently, there are various types of transgenic technologies, including the microinjection method, sperm carrier method, somatic cell cloning method, retrovirus method, embryonic stem cell-mediated method and oocyte carrier method [4,5]. The microinjection technology microinjects exogenous DNA into the former nucleus of a fertilized egg cell by a microscopic instrument [4].…”

“…The microinjection technology microinjects exogenous DNA into the former nucleus of a fertilized egg cell by a microscopic instrument [4]. The sperm carrier method incubates the exogenous gene with the live sperm, and then, the live sperm will take up the exogenous genes and be fertilized in vitro; finally, the transgenic mice model will be acquired [6,7].…”