Mature seed-derived callus of the model indica rice variety Kasalath is highly competent in Agrobacterium-mediated transformation

We previously established an efficient Agrobacterium-mediated transformation system using primary calli derived from mature seeds of the model japonica rice variety Nipponbare. We expected that the shortened tissue culture period would reduce callus browning—a common problem with the indica transformation system during prolonged tissue culture in the undifferentiated state. In this study, we successfully applied our efficient transformation system to Kasalath—a model variety of indica rice. The Luc reporter system is sensitive enough to allow quantitative analysis of the competency of rice callus for Agrobacterium-mediated transformation. We unexpectedly discovered that primary callus of Kasalath exhibits a remarkably high competency for Agrobacterium-mediated transformation compared to Nipponbare. Southern blot analysis and Luc luminescence showed that independent transformation events in primary callus of Kasalath occurred successfully at ca. tenfold higher frequency than in Nipponbare, and single copy T-DNA integration was observed in ~40% of these events. We also compared the competency of secondary callus of Nipponbare and Kasalath and again found superior competency in Kasalath, although the identification and subsequent observation of independent transformation events in secondary callus is difficult due to the vigorous growth of both transformed and non-transformed cells. An efficient transformation system in Kasalath could facilitate the identification of QTL genes, since many QTL genes are analyzed in a Nipponbare × Kasalath genetic background. The higher transformation competency of Kasalath could be a useful trait in the establishment of highly efficient systems involving new transformation technologies such as gene targeting

Changes in Primary and Secondary Metabolite Levels in Response to Gene Targeting-Mediated Site-Directed Mutagenesis of the Anthranilate Synthase Gene in Rice

Gene targeting (GT) via homologous recombination allows precise modification of a target gene of interest. In a previous study, we successfully used GT to produce rice plants accumulating high levels of free tryptophan (Trp) in mature seeds and young leaves via targeted modification of a gene encoding anthranilate synthase—a key enzyme of Trp biosynthesis. Here, we performed metabolome analysis in the leaves and mature seeds of GT plants. Of 72 metabolites detected in both organs, a total of 13, including Trp, involved in amino acid metabolism, accumulated to levels >1.5-fold higher than controls in both leaves and mature seeds of GT plants. Surprisingly, the contents of certain metabolites valuable for both humans and livestock, such as γ-aminobutyric acid and vitamin B, were significantly increased in mature seeds of GT plants. Moreover, untargeted analysis using LC-MS revealed that secondary metabolites, including an indole alkaloid, 2-[2-hydroxy-3-β-d-glucopyranosyloxy-1-(1H-indol-3-yl)propyl] tryptophan, also accumulate to higher levels in GT plants. Some of these metabolite changes in plants produced via GT are similar to those observed in plants over expressing mutated genes, thus demonstrating that in vivo protein engineering via GT can be an effective approach to metabolic engineering in crops

A mouse model of glottal closure for the treatment of breathlessness and hoarseness

　嗄声は，炎症や腫瘍性病変以外にも老化や筋疾患に伴う声帯筋萎縮や反回神経麻痺などに よって発症する．うまく発声できないことはコミュニケーション能力の極端な低下を意味し， Quality of life（QOL）を著しく低下させる．その病態は疾患によって異なるが，声門閉鎖不全に より発声時に生じる声門間隙の残存があると気息性嗄声を生じる．気息性嗄声の治療は，保存的治 療から外科治療まで様々なものが存在するが専門性が高く限られた施設のみで治療される現状で， 治療を受けられる患者が限定される問題がある．そのためさらに低侵襲で復元性の高い治療や汎用性の高い治療が望まれる．再生医療をふくめた新規治療の開発を進めるためには，まず声門閉鎖不全を生ずるマウスモデルの確立が必要である． 　本研究では，C57BL/6マウスを用いて神経原性の声門閉鎖不全モデルとしての反回神経麻痺モ デルと，加齢性の声門閉鎖不全モデルの２つのモデル動物を作成し，内喉頭筋を含む声帯の評価を行うことでマウスにおける声門閉鎖不全のメカニズムがヒトと同一であるかどうかを解明することを目的とした．また筋肉の過形成を抑制する分子である Myostatin（以下，Mstn）を標的とし，変 異 Mstn を過剰発現することで全身性に筋過形成を来すマウス（以下，変異 Mstn tg マウス）を用いて内喉頭筋を含む声帯の評価を行い，Mstn 阻害の臨床応用の可能性についても検討した．　反回神経麻痺モデルマウスについては，健側と比較して麻痺側では有意に甲状披裂筋萎縮が認められ，声門閉鎖不全のメカニズムはヒトと同一でありモデルとして有効であると考えられた．加齢マウスを用いた検討では，内視鏡で，声帯萎縮と弓状変化を確認出来た．甲状披裂筋萎縮はみられず声帯粘膜の萎縮とコラーゲン線維の増加が確認され，加齢に伴うヒト声帯の萎縮と同様の形態学的所見を呈することから加齢に伴う声門閉鎖不全のモデルとして有用であると考えた．変異 Mstn tg マウスの内喉頭筋においては有意な筋肉量・筋線維の増大はなかったが，傍声帯間隙の脂肪が減少しており，喉頭内においても脂肪代謝に影響があることが明らかになった．　In addition to inflammatory and neoplastic lesions, hoarseness can also be caused by vocal fold muscle atrophy and recurrent nerve palsy associated with aging and muscle diseases. The inability to speak well leads to a drastic decrease in communication ability and quality of life (QOL). The pathogenesis differs depending on the disease, but the residual glottal gap caused by inadequate glottal closure during vocalization results in hoarseness. There are various treatments for hoarseness, ranging from conservative to surgical treatment, but they are highly specialized and are only available at limited facilities, limiting the number of patients who can receive treatment. Therefore, minimally invasive, highly restorative, and highly versatile treatments are desired. To develop new therapies, including regenerative medicine, the establishment of a mouse model of glottic atresia is necessary.　In this study, we created two models of glottic insufficiency in C57BL/6 mice: a neurogenic model of recurrent nerve palsy and an age-related model of glottic insufficiency. The purpose of this study was to clarify whether the mechanism of glottal closure in mice is the same as that in humans. In addition, we targeted myostatin (Mstn), a molecule that inhibits muscle hyperplasia, and evaluated the vocal folds, including the internal laryngeal muscle, in mice overexpressing mutant Mstn (mutant Mstn tg mice). This mutation causes systemic muscle hyperplasia and allows investigation of the possible clinical application of Mstn inhibition, which was also examined.　In the mouse model of recurrent nerve palsy, significant atrophy of the lamina propria muscle was observed on the paralyzed side, compared with the healthy side, suggesting that the mechanism of glottic insufficiency is the same as in humans and that this would be an effective model. The mechanism of glottic atrophy in mice is the same as that in humans, also making it a useful model. In the case of mutant Mstn tg mice, there was no significant increase in muscle mass or muscle fiber numbers in the endolaryngeal muscle. Further, in the endolaryngeal muscles, there was no significant increase in muscle mass or muscle fiber numbers; however, there was decreased fat in the paralaryngeal gap, indicating that laryngeal fat metabolism is also affected