原 弘真

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.

We conducted two lines of genome-editing experiments of mouse hematopoietic stem cells (HSCs) with the clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9). First, to evaluate the genome-editing efficiency in mouse bona fide HSCs, we knocked out integrin alpha 2b (Itga2b) with Cas9 ribonucleoprotein (Cas9/RNP) and performed serial transplantation in mice. The knockout efficiency was estimated at approximately 15%. Second, giving an example of X-linked severe combined immunodeficiency (X-SCID) as a target genetic disease, we showed a proof-of-concept of universal gene correction, allowing rescue of most of X-SCID mutations, in a completely non-viral setting. We inserted partial cDNA of interleukin-2 receptor gamma chain (Il2rg) into intron 1 of Il2rg via non-homologous end-joining (NHEJ) with Cas9/RNP and a homology-independent targeted integration (HITI)-based construct. Repaired HSCs reconstituted T lymphocytes and thymuses in SCID mice. Our results show that a non-viral genome-editing of HSCs with CRISPR/Cas9 will help cure genetic diseases.

A knock-in can generate fluorescent or Cre-reporter under the control of an endogenous promoter. It also generates knock-out or tagged-protein with fluorescent protein and short tags for tracking and purification. Recent advances in genome editing with clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9) significantly increased the efficiencies of making knock-in cells. Here we describe the detailed protocols of generating knock-in mouse and human pluripotent stem cells (PSCs) by electroporation and lipofection, respectively.

We report that a sheep fetal liver provides a microenvironment for generating hematopoietic cells with long-term engrafting capacity and multilineage differentiation potential from human induced pluripotent stem cell (iPSC)-derived hemogenic endothelial cells (HEs). Despite the promise of iPSCs for making any cell types, generating hematopoietic stem and progenitor cells (HSPCs) is still a challenge. We hypothesized that the hematopoietic microenvironment, which exists in fetal liver but is lacking in vitro, turns iPSC-HEs into HSPCs. To test this, we transplanted CD45-negative iPSC-HEs into fetal sheep liver, in which HSPCs first grow. Within 2 months, the transplanted cells became CD45 positive and differentiated into multilineage blood cells in the fetal liver. Then, CD45-positive cells translocated to the bone marrow and were maintained there for 3 years with the capability of multilineage differentiation, indicating that hematopoietic cells with long-term engraftment potential were generated. Moreover, human hematopoietic cells were temporally enriched by xenogeneic donor-lymphocyte infusion into the sheep. This study could serve as a foundation to generate HSPCs from iPSCs.

We have proposed a new hypothesis for cryodamage of bovine oocytes; multiple aster formation frequently observed in vitrified-warmed and fertilized oocytes may be related to loss of ooplasmic function responsible for normal microtubule assembly from sperm-aster. The present study was conducted to investigate whether recovery culture of vitrified-warmed bovine oocytes with an inhibitor (Y-27632) of Rho-associated coiled-coil kinase (ROCK) can improve the developmental potential after IVF and IVC. Cumulus-free bovine IVM oocytes were subjected to Cryotop vitrification. Treatment of the post-warm oocytes with 10 μM Y-27632 for 2 h resulted in the significantly higher oocyte survival rate prior to the IVF (97.8 vs 90.2%), cleavage rate (72.4 vs 56.2%) and Day-8 blastocyst formation rate (21.4 vs 13.9%). Time-dependent change in mitochondrial activity of the vitrified-warmed oocytes was not influenced by the ROCK inhibition during the period of recovery culture. Also, kinetics of actin filaments until 10 hpi was not affected by the Y-27632 treatment. At the 10 hpi, proportions of oocytes formed aster(s) were comparable (80.7 and 81.8%), but the ability of ooplasm to support single-aster formation was improved by the ROCK inhibition (67.1 vs 49.4%). In conclusion, inhibition of ROCK activity in vitrified-warmed bovine oocytes during a short-term recovery culture can lead to the higher developmental competence, probably due to decreased apoptosis and normalized function of microtubule-organizing center.

【目的】精子の凍結保存に代わる保存方法として凍結乾燥が注目されているが，満足のいく発生成績が報告されている動物種は限られている。凍結乾燥を適切な条件下で行うと溶液は多孔質のスポンジ様構造 (凍結乾燥ケーキ) を形成する。しかし，溶液の最大凍結濃縮相ガラス転移温度 (Tg') よりも高い温度で乾燥を行うと，コラプスと呼ばれる凍結乾燥ケーキの構造崩壊が生じやすい。本研究では，凍結乾燥ケーキに生じたコラプスが凍結乾燥ウシ精子の発生能に及ぼす影響について調べた。【方法と結果】示差走査熱量計を用いて測定した，10 mM Tris-HCl，50 mM EGTA，0.5 M トレハロースからなる凍結乾燥バッファーのTg'は，−28℃だった。次に，同バッファーに懸濁した凍結融解ウシ精子を予備凍結したのち，−30℃または0℃に制御した凍結乾燥機中で6時間の乾燥を行った。乾燥温度が−30℃の凍結乾燥ケーキは完全な形状を保っていた一方，Tg'より高い0℃で乾燥した場合にはコラプスが生じていた。続いて，得られた凍結乾燥ケーキの乾燥程度を調べるために含水率およびガラス転移温度 (Tg) の測定を行った。0℃で乾燥した場合の含水率は−30℃の場合と比べて約5倍 (3.6 vs. 0.7%, P < 0.05) だったが，Tgの測定によりいずれも十分な乾燥状態にあることが確認できた。−20℃で一晩保存した凍結乾燥精子を顕微授精 (ICSI) に供したところ，卵割率に差は認められなかった。しかし，胚盤胞発生率については0℃で乾燥した場合の値 (1%：卵割ベース) が−30℃で乾燥したときの値 (14%) よりも有意 (P < 0.05) に低下した。現在さらに，凍結乾燥精子の微細構造やDNA損傷に対する影響を調べている。以上，凍結乾燥ケーキのコラプスはウシ精子に対する乾燥ストレスを増加させ，ICSI後の胚盤胞発生能を低下させることが明らかとなった。

【目的】ガラス化・加温したウシ卵子では体外受精後に複数の星状体が出現する例が多発し，これが一因となって胚盤胞発生率が低下している可能性がある (Theriogenology 2012; 77: 908-15)。また，ウシ精子中心体のMTOC機能はグルタチオン合成阻害剤による卵子処理で損なわれるという報告がある。本研究ではウシ卵子の体外成熟行程において細胞内グルタチオンレベルの上昇が期待できる薬剤処理を行い，ガラス化・体外受精後の精子星状体形成ならびに胚盤胞発生との関係を調べた。【方法】屠殺の翌日まで約10℃で保存したウシ卵巣から卵丘卵母細胞複合体を回収し，&beta;-メルカプトエタノール (&beta;-ME) とL-システイン (L-Cys) を添加した成熟培地で22時間培養した。裸化後に成熟卵子率を求め，クライオトップをデバイスとしてガラス化保存した。加温卵子のグルタチオンレベルはDTNB-グルタチオン還元酵素リサイクリングアッセイにより，体外受精10時間目の星状体形成率と複数星状体の発生頻度は&alpha;チューブリンに対する免疫染色により，そして胚盤胞発生率は低酸素濃度下での体外培養により求めた。【結果】成熟卵子率に&beta;-ME / L-Cysの添加による影響はなかったが，無処理対照区よりも細胞内グルタチオンレベルは約3倍に上昇しており，このレベルはガラス化行程が加わっても下がることはなかった。精子星状体形成率は実験区に関わらず，いずれも95%以上だった。これらの星状体形成卵子のうち複数の精子星状体が観察された卵子の割合はガラス化区で新鮮対照区よりも3〜4倍多くなっており，&beta;-ME / L-Cys添加によって細胞内グルタチオンレベルが高まっている場合でも同様であった。胚盤胞発生率においても&beta;-ME / L-Cys添加の有無に関わらず，ガラス化区と新鮮対照区のあいだで2倍強の差が認められた。以上の結果から，ガラス化・加温卵子におけるグルタチオンレベルと胚盤胞発生率との因果関係の存在について否定的な見解に達した。