分子病態治療研究センター 再生医学研究部

谷原 史倫

Fuminori Tanihara

基本情報

所属
自治医科大学 先端医療技術開発センター 動物資源ラボラトリー 准教授
学位
博士(獣医学)(2014年3月 山口大学)

研究者番号
90754680
J-GLOBAL ID
201801008049373758
researchmap会員ID
B000318119

動物の発生工学、生殖工学分野で、ブタの体外胚生産技術やゲノム編集に関する研究を行っています。

研究キーワード

 4

論文

 102
  • Fuminori Tanihara, Maki Hirata, Zhao Namula, Manita Wittayarat, Lanh Thi Kim Do, Qingyi Lin, Koki Takebayashi, Hiromasa Hara, Megumi Nagahara, Takeshige Otoi
    Molecular biology reports 50(6) 5049-5057 2023年6月  査読有り筆頭著者
    BACKGROUND: Pigs are excellent large animal models with several similarities to humans. They provide valuable insights into biomedical research that are otherwise difficult to obtain from rodent models. However, even if miniature pig strains are used, their large stature compared with other experimental animals requires a specific maintenance facility which greatly limits their usage as animal models. Deficiency of growth hormone receptor (GHR) function causes small stature phenotypes. The establishment of miniature pig strains via GHR modification will enhance their usage as animal models. Microminipig is an incredibly small miniature pig strain developed in Japan. In this study, we generated a GHR mutant pig using electroporation-mediated introduction of the CRISPR/Cas9 system into porcine zygotes derived from domestic porcine oocytes and microminipig spermatozoa. METHODS AND RESULTS: First, we optimized the efficiency of five guide RNAs (gRNAs) designed to target GHR in zygotes. Embryos that had been electroporated with the optimized gRNAs and Cas9 were then transferred into recipient gilts. After embryo transfer, 10 piglets were delivered, and one carried a biallelic mutation in the GHR target region. The GHR biallelic mutant showed a remarkable growth-retardation phenotype. Furthermore, we obtained F1 pigs derived from the mating of GHR biallelic mutant with wild-type microminipig, and GHR biallelic mutant F2 pigs through sib-mating of F1 pigs. CONCLUSIONS: We have successfully demonstrated the generation of biallelic GHR-mutant small-stature pigs. Backcrossing of GHR-deficient pig with microminipig will establish the smallest pig strain which can contribute significantly to the field of biomedical research.
  • Thanh Van Nguyen, Lanh Thi Kim Do, Zhao Namula, Qingyi Lin, Nanaka Torigoe, Megumi Nagahara, Maki Hirata, Fuminori Tanihara, Takeshige Otoi
    Cryo-Letters 44(2) 118-122 2023年3月  査読有り
    BACKGROUND: Cryopreservation of bovine zygotes allows for a flexible schedule of genome editing via electroporation. However, vitrification-induced cell membrane damage may not only affect embryonic development but also genome mutation. OBJECTIVE: To investigate the effects of vitrification of zygotes before and after electroporation treatments on the development and genome mutation of bovine presumptive zygotes. MATERIALS AND METHODS: In vitro-derived bovine zygotes were electroporated with the CRISPR/Cas9 system immediately (Vitrified-EP) or 2 h after incubation (Vitrified-2h-EP) following vitrification and warming, or electroporated before vitrification (EP-vitrified). RESULTS: The development rates of vitrified-warmed zygotes were significantly lower (p < 0.05) than those of control zygotes that were not vitrified. Moreover, no differences were observed in the mutation rates and mutation efficiency of the blastocysts resulting from electroporated zygotes, irrespective of the timing of electroporation treatment. CONCLUSION: Our results suggest that vitrification before and after electroporation treatments does not affect the genome editing of zygotes.
  • Chommanart Thongkittidilok, Maki Hirata, Qingyi Lin, Nanaka Torigoe, Bin Liu, Yoko Sato, Megumi Nagahara, Fuminori Tanihara, Takeshige Otoi
    Veterinary medicine international 2023 7000858-7000858 2023年  
    Pigs rarely develop cancer; however, tumour protein p53 (TP53)-modified pigs may have an increased incidence of cancer. In this study, two pigs with mosaic mutations induced by gene editing were compared to determine the role of the wild-type TP53 sequence in tumorigenesis and to speculate how amino acid changes in TP53 sequences are related to tumorigenesis. The pig without tumours had a wild-type TP53 sequence and a 1-bp deletion in the TP53 sequence that resulted in a premature stop codon. In contrast, the pig with nephroblastoma had 6- and 7-bp deletions in the TP53 sequence, resulting in the absence of two amino acids and a premature stop codon, respectively. Our results indicated that TP53 mutations with truncated amino acids may be related to tumour formation.
  • Fuminori Tanihara, Maki Hirata, Zhao Namula, Lanh Thi Kim Do, Naoaki Yoshimura, Qingyi Lin, Koki Takebayashi, Tetsushi Sakuma, Takashi Yamamoto, Takeshige Otoi
    Frontiers in cell and developmental biology 11 884340 2023年  査読有り筆頭著者
    Just one amino acid at the carboxy-terminus of the B chain distinguishes human insulin from porcine insulin. By introducing a precise point mutation into the porcine insulin (INS) gene, we were able to generate genetically modified pigs that secreted human insulin; these pigs may be suitable donors for islet xenotransplantation. The electroporation of the CRISPR/Cas9 gene-editing system into zygotes is frequently used to establish genetically modified rodents, as it requires less time and no micromanipulation. However, electroporation has not been used to generate point-mutated pigs yet. In the present study, we introduced a point mutation into porcine zygotes via electroporation using the CRISPR/Cas9 system to generate INS point-mutated pigs as suitable islet donors. We first optimized the efficiency of introducing point mutations by evaluating the effect of Scr7 and the homology arm length of ssODN on improving homology-directed repair-mediated gene modification. Subsequently, we prepared electroporated zygotes under optimized conditions and transferred them to recipient gilts. Two recipients became pregnant and delivered five piglets. Three of the five piglets carried only the biallelic frame-shift mutation in the INS gene, whereas the other two successfully carried the desired point mutation. One of the two pigs mated with a WT boar, and this desired point mutation was successfully inherited in the next F1 generation. In conclusion, we successfully established genetically engineered pigs with the desired point mutation via electroporation-mediated introduction of the CRISPR/Cas9 system into zygotes, thereby avoiding the time-consuming and complicated micromanipulation method.
  • Fuminori Tanihara, Maki Hirata, Takeshige Otoi
    Methods in molecular biology (Clifton, N.J.) 2637 293-300 2023年  招待有り筆頭著者
    Pigs are excellent large animal models owing to their several physiological and anatomical similarities to humans. Somatic cell nuclear transfer using gene-modified cells is the mainstream approach for generating genetically modified pigs. Recent advances in improving gene editors such as the CRISPR/Cas9 system have enabled direct gene modification in zygotes/embryos. Here, we describe the gene editing by electroporation of Cas9 protein (GEEP) method, an optimized electroporation-mediated method for the introduction of CRISPR/Cas9 into porcine zygotes/embryos. The simplicity and micromanipulation-free procedures are the major advantages of this method.

MISC

 7

共同研究・競争的資金等の研究課題

 8

産業財産権

 1