Year: 2018
Type: article
Abstract: Animal genome engineering experimental procedures involve three major steps: isolation of zygotes from pregnant females; microinjection of zygotes, and; transfer of injected zygotes into recipient females, that have been practiced for over three decades. The laboratory set ups intending to performing these procedures require to have sophisticated equipment as well as highly skilled technical personnel. Because of these reasons, animal transgenesis experiments are typically performed at centralized core facilities in most research organizations. We recently showed that all three steps, of animal transgensis, can be bypassed using a method termed GONAD ( G enome-editing via O viductal N ucleic A cids D elivery), by directly electroporating genome editing components into zygotes in situ . Although our first report demonstrated the genome-editing capability, its efficiency was lower than the standard methods using microinjection. Here we investigated critical parameters of GONAD to make it suitable for creating animal models of large genomic deletions, single nucleotide corrections and long sequence insertions. The efficiency of genome editing in the improved GONAD ( i -GONAD) method reached to the levels comparable to traditional microinjection methods. The streamlined parameters, and the simplified experimental steps, in the i -GONAD method makes it suitable for routine genome editing applications performed both at centralized facilities as well as at the laboratories that lack highly skilled personnel and the sophisticated equipment.
Source: Genome biology
Institutions: Tokai University, Institute of Medical Sciences, Tokai University, Kagoshima University, Hamamatsu University, Hamamatsu University School of Medicine, Shigei Medical Research Institute, National Defense Medical College, Tokyo University of Agriculture, University of Nebraska Medical Center
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $5,490
Awards: Easi-CRISPR for creating genetically modified mice using long ssDNA., G1642921554, Development of gene transfer to peripheral tissues surrounding tooth (GTPT) for the tissue around the root forming apex of juvenile mice and its application, G2234251972, Development of novel method for generation of genome editing mice without ex vivo handling of embryos, Development and application of liver-specific delivery carrier made by self-aggregate, G3514114876, Efficient enrichment of homozygous bi-allelic knockout microminiature porcine cells using a novel selection system and production of genome-edited cloned piglets, Analysis of Amyotrophic lateral sclerosis using Chmp2A / B mutant mice, Development of microinjection-independent animal genome manipulation method
OpenAlex ID: W2796841946