Target-selective homologous recombination cloning for high-throughput generation of monoclonal antibodies from single plasma cells

<p>Abstract</p> <p>Background</p> <p>Molecular cloning of functional immunoglobulin genes from single plasma cells is one of the most promising technologies for the rapid development of monoclonal antibody drugs. However, the proper insertion of PCR-amplified immunoglobulin genes into expression vectors remains an obstacle to the high-throughput production of recombinant monoclonal antibodies.</p> <p>Results</p> <p>We developed a single-step cloning method, target-selective homologous recombination (TS-HR), in which PCR-amplified immunoglobulin variable genes were selectively inserted into vectors, even in the presence of nonspecifically amplified DNA. TS-HR utilizes Red/ET-mediated homologous recombination with a target-selective vector (TS-vector) with unique homology arms on its termini. Using TS-HR, immunoglobulin variable genes were cloned directly into expression vectors by co-transforming unpurified PCR products and the TS-vector into <it>E. coli</it>. Furthermore, the high cloning specificity of TS-HR allowed plasmids to be extracted from pools of transformed bacteria without screening single colonies for correct clones. We present a one-week protocol for the production of recombinant mouse monoclonal antibodies from large numbers of single plasma cells.</p> <p>Conclusion</p> <p>The time requirements and limitations of traditional cloning procedures for the production of recombinant immunoglobulins have been significantly reduced with the development of the TS-HR cloning technique.</p

Collagen-VI supplementation by cell transplantation improves muscle regeneration in Ullrich congenital muscular dystrophy model mice

6型コラーゲンを補う細胞移植がウルリッヒ型先天性筋ジストロフィーモデルマウスの病態を改善する. 京都大学プレスリリース. 2021-08-24.iPS cells show therapeutic benefits for a rare muscle dystrophy. 京都大学プレスリリース. 2021-08-24.[Background] Mesenchymal stromal cells (MSCs) function as supportive cells on skeletal muscle homeostasis through several secretory factors including type 6 collagen (COL6). Several mutations of COL6A1, 2, and 3 genes cause Ullrich congenital muscular dystrophy (UCMD). Skeletal muscle regeneration deficiency has been reported as a characteristic phenotype in muscle biopsy samples of human UCMD patients and UCMD model mice. However, little is known about the COL6-dependent mechanism for the occurrence and progression of the deficiency. The purpose of this study was to clarify the pathological mechanism of UCMD by supplementing COL6 through cell transplantation. [Methods] To test whether COL6 supplementation has a therapeutic effect for UCMD, in vivo and in vitro experiments were conducted using four types of MSCs: (1) healthy donors derived-primary MSCs (pMSCs), (2) MSCs derived from healthy donor induced pluripotent stem cell (iMSCs), (3) COL6-knockout iMSCs (COL6KO-iMSCs), and (4) UCMD patient-derived iMSCs (UCMD-iMSCs). [Results] All four MSC types could engraft for at least 12 weeks when transplanted into the tibialis anterior muscles of immunodeficient UCMD model (Col6a1KO) mice. COL6 protein was restored by the MSC transplantation if the MSCs were not COL6-deficient (types 1 and 2). Moreover, muscle regeneration and maturation in Col6a1KO mice were promoted with the transplantation of the COL6-producing MSCs only in the region supplemented with COL6. Skeletal muscle satellite cells derived from UCMD model mice (Col6a1KO-MuSCs) co-cultured with type 1 or 2 MSCs showed improved proliferation, differentiation, and maturation, whereas those co-cultured with type 3 or 4 MSCs did not. [Conclusions] These findings indicate that COL6 supplementation improves muscle regeneration and maturation in UCMD model mice

小学校給食で提供されるエネルギーおよび主要栄養素量の妥当性についての検討

We performed food consumption survey in July, 2007 for the 2nd and 5th grade children at an elementary school of Nagano city to evaluate the amounts of energy and nutrients offered with school lunch. Energy intake of the 2nd grade was comparable to the estimated energy requirement (EER), while that of the 5th grade was greatly lower than the EER. Most of nutrients, except iron and dietary fiber, were within the recommendable range. As to the consumption ratio from each meal, energy, protein, lipid and iron showed the similar pattern, being about 33% of total daily consumption from lunch. As for daily consumption of calcium and vitamin B2, intake ratio from lunch was about 50% and 40%, respectively. Results confirmed the important role of school lunch service to compensate the amounts of nutrients which are easy to lack in daily food consumption

Relationship between Protection against Cold and the Physiological Index during a Cold Environment

A snow cave is a bivouac shelter used in mountain climbing that is widely used as a shelter against the cold during winter. In the outdoors, wind velocity and air temperature have an influence on temperature change. It could stabilize body temperature if it can control the convection of ambient air. This paper could develop a theory focusing on the relation between physiological indexes and the protection against the cold while staying in a snow cave. For example, protection against the cold could be thermal insulation underwear, thermal insulation gloves, thermal insulation socks, a steam warmed temperature sheet and a rescue sheet. Measurement items were heart rate, blood pressure, rectal temperature, score of a subjective thermal sensation and the activity of the parasympathetic nervous system. It was clarified that the protection against the cold could be effective for the decrease of the physiological index. These field studies suggest that they would enable the adaptation in the adjustment range of the autonomic nervous system given these protections against the cold

平成29年度「卒業研究」実践報告

今年度の卒業研究はテーマ設定に関する時間を確保するために、2年次の「T-GAP」の内容に卒業研究の内容を含めて実施した。評価に関しては国際バカロレアの「課題論文」(EE)を参考にしつつ、生徒の実態に沿う形で評価規準を作り直して使用した。テーマ設定に時間をかけたころとなどにより、多くの先行研究を参考にし、主体的に課題を設定して卒業研究に取り組む姿が確認された

Rapid production of antigen-specific monoclonal antibodies from a variety of animals

<p>Abstract</p> <p>Background</p> <p>Although a variety of animals have been used to produce polyclonal antibodies against antigens, the production of antigen-specific monoclonal antibodies from animals remains challenging.</p> <p>Results</p> <p>We propose a simple and rapid strategy to produce monoclonal antibodies from a variety of animals. By staining lymph node cells with an antibody against immunoglobulin and a fluorescent dye specific for the endoplasmic reticulum, plasma/plasmablast cells were identified without using a series of antibodies against lineage markers. By using a fluorescently labeled antigen as a tag for a complementary cell surface immunoglobulin, antigen-specific plasma/plasmablast cells were sorted from the rest of the cell population by fluorescence-activated cell sorting. Amplification of cognate pairs of immunoglobulin heavy and light chain genes followed by DNA transfection into 293FT cells resulted in the highly efficient production of antigen-specific monoclonal antibodies from a variety of immunized animals.</p> <p>Conclusions</p> <p>Our technology eliminates the need for both cell propagation and screening processes, offering a significant advantage over hybridoma and display strategies.</p

LXXLL-Related Motifs in Dax-1 Have Target Specificity for the Orphan Nuclear Receptors Ad4BP/SF-1 and LRH-1

The orphan receptor Ad4BP/SF-1 (NR5A1) is a constitutive activator, and its activity is repressed by another orphan receptor, Dax-1 (NR0B1). In the present study, we investigated the molecular mechanisms underlying this repression by Dax-1. Yeast two-hybrid and transient-transfection assays confirmed the necessity of three LXXLL-related motifs in Dax-1 for interaction with and repression of Ad4BP/SF-1. In vitro pull-down experiments confirmed that Dax-1 interacts with Ad4BP/SF-1 and also with LRH-1 (NR5A2). The target specificity of the LXXLL-related motifs was indicated by the observations that Ad4BP/SF-1, ERα (NR3A1), LRH-1, ERR2 (NR3B2), and fly FTZ-F1 (NR5A3) interacted through their ligand binding domains with all the LXXLL-related motifs in Dax-1 whereas HNF4 (NR2A1) and RORα (NR1F1) did not. Transcriptional activities of the receptors whose DNA binding domains (DBDs) were replaced by the GAL4 DBD were repressed by Dax-1 to various levels, which correlated with the strength of interaction. Amino acid substitutions revealed that Ad4BP/SF-1 and LRH-1 preferentially interact with L(+1)XXLL-related motifs containing serine, tyrosine, serine, and threonine at positions −2, +2, +3, and +6, respectively. Taken together, our results indicate that the specificities of LXXLL-related motifs in Dax-1 based on their amino acid sequences play an important role in regulation of orphan receptors