レガシー ソフトウェア ニ オケル フォールト ハッセイ ヨソク ノ タメノ ヒセンケイ ハンベツ ブンセキホウ

http://library.naist.jp/mylimedio/dllimedio/show.cgi?bookid=100020305&oldid=28107修士 (Master)工学 (Engineering)修第1286

Recent Advances and Future Perspectives of In Vivo Targeted Delivery of Genome-Editing Reagents to Germ cells, Embryos, and Fetuses in Mice

The recently discovered clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) systems that occur in nature as microbial adaptive immune systems are considered an important tool in assessing the function of genes of interest in various biological systems. Thus, development of efficient and simple methods to produce genome-edited (GE) animals would accelerate research in this field. The CRISPR/Cas9 system was initially employed in early embryos, utilizing classical gene delivery methods such as microinjection or electroporation, which required ex vivo handling of zygotes before transfer to recipients. Recently, novel in vivo methods such as genome editing via oviductal nucleic acid delivery (GONAD), improved GONAD (i-GONAD), or transplacental gene delivery for acquiring genome-edited fetuses (TPGD-GEF), which facilitate easy embryo manipulation, have been established. Studies utilizing these techniques employed pregnant female mice for direct introduction of the genome-editing components into the oviduct or were dependent on delivery via tail-vein injection. In mice, embryogenesis occurs within the oviducts and the uterus, which often hampers the genetic manipulation of embryos, especially those at early postimplantation stages (days 6 to 8), owing to a thick surrounding layer of tissue called decidua. In this review, we have surveyed the recent achievements in the production of GE mice and have outlined the advantages and disadvantages of the process. We have also referred to the past achievements in gene delivery to early postimplantation stage embryos and germ cells such as primordial germ cells and spermatogonial stem cells, which will benefit relevant research

Generation of Flag/DYKDDDDK Epitope Tag Knock-In Mice Using i-GONAD Enables Detection of Endogenous CaMKIIα and β Proteins

Specific antibodies are necessary for cellular and tissue expression, biochemical, and functional analyses of protein complexes. However, generating a specific antibody is often time-consuming and effort-intensive. The epitope tagging of an endogenous protein at an appropriate position can overcome this problem. Here, we investigated epitope tag position using AlphaFold2 protein structure prediction and developed Flag/DYKDDDDK tag knock-in CaMKIIα and CaMKIIβ mice by combining CRISPR-Cas9 genome editing with electroporation (i-GONAD). With i-GONAD, it is possible to insert a small fragment of up to 200 bp into the genome of the target gene, enabling efficient and convenient tagging of a small epitope. Experiments with commercially available anti-Flag antibodies could readily detect endogenous CaMKIIα and β proteins by Western blotting, immunoprecipitation, and immunohistochemistry. Our data demonstrated that the generation of Flag/DYKDDDDK tag knock-in mice by i-GONAD is a useful and convenient choice, especially if specific antibodies are unavailable

Phenotype-Based Search of Natural Mutations Related to Hereditary Diseases Existing in a Closed Colony of Mice

We attempted to detect natural mutations existing in the Jcl:ICR closed colony of mice which is maintained by random mating. We used ordinary genetic backcrosses to efficiently detect recessive mutations carried by individual mice in the colony. Crosses of DBA/2 females and ICR males were performed to obtain F(1) mice. Four F(1) females randomly selected from each cross were backcrossed to the male parent. More than thirty backcross progeny were obtained from each F(1) female by several deliveries. Phenotypes of the backcross progeny were observed macroscopically at about one month of age. As a result, 18 (26.1%) of 69 Jcl:ICR males carried 11 recessive mutation(s). Based on the phenotypes, the tentative names were abnormal kidney, aplasia of eyelids/hind limb digits, circling, dwarfism, heterotaxy, hind limb paralysis, hydrocephalus, rigidity (or rigor), testicular hypoplasia, tremor, and wobbling. The genes responsible for aplasia of eyelids/hind limb digits and dwarfism were each carried by two males, the genes responsible for hydrocephalus and testicular hypoplasia were each carried by three males and the gene responsible for wobbling by four males. It was strongly suggested that the genes shared by several males originated from an identical mutated gene. Surprisingly, male No. 43 had the responsible genes of abnormal kidneys and testicular hypoplasia, and No. 79 had those of dwarfism and tremor. The results obtained in this study suggest that breeders need to be aware of the presence of natural mutations in their colonies