Targeted mutagenesis in chicken using CRISPR/Cas9 system

The CRISPR/Cas9 system is a simple and powerful tool for genome editing in various organisms including livestock animals. However, the system has not been applied to poultry because of the difficulty in accessing their zygotes. Here we report the implementation of CRISPR/Cas9-mediated gene targeting in chickens. Two egg white genes, ovalbumin and ovomucoid, were efficiently (> 90%) mutagenized in cultured chicken primordial germ cells (PGCs) by transfection of circular plasmids encoding Cas9, a single guide RNA, and a gene encoding drug resistance, followed by transient antibiotic selection. We transplanted CRISPR-induced mutant-ovomucoid PGCs into recipient chicken embryos and established three germline chimeric roosters (G0). All of the roosters had donor-derived mutant-ovomucoid spermatozoa, and the two with a high transmission rate of donor-derived gametes produced heterozygous mutant ovomucoid chickens as about half of their donor-derived offspring in the next generation (G1). Furthermore, we generated ovomucoid homozygous mutant offspring (G2) by crossing the G1 mutant chickens. Taken together, these results demonstrate that the CRISPR/Cas9 system is a simple and effective gene-targeting method in chickens.ArticleSCIENTIFIC REPORTS.6:23980(2016)journal articl

Cre-LoxP-regulated expression of monoclonal antibodies driven by an ovalbumin promoter in primary oviduct cells

<p>Abstract</p> <p>Background</p> <p>A promoter capable of driving high-level transgene expression in oviduct cells is important for developing transgenic chickens capable of producing therapeutic proteins, including monoclonal antibodies (mAbs), in the whites of laid eggs. Ovalbumin promoters can be used as oviduct-specific regulatory sequences in transgenic chickens, but their promoter activities are not high, according to previous reports.</p> <p>Results</p> <p>In this study, while using a previously characterized ovalbumin promoter, we attempted to improve the expression level of mAbs using a Cre/<it>lox</it>P-mediated conditional excision system. We constructed a therapeutic mAb expression vector, pBS-DS-hIgG, driven by the CMV and CAG promoters, in which the expression of the heavy and light chains of humanized immunoglobulin G (hIgG) is preceded by two floxed stuffer reporter genes. In the presence of Cre, the stuffer genes were precisely excised and hIgG expression was induced in pBS-DS-hIgG-transfected 293T cells. In chicken oviduct primary culture cells, hIgG was expressed after transfection of pBS-DS-hIgG together with the ovalbumin promoter-driven Cre expression vector. The expression level of hIgG in these cells was increased 40-fold over that induced directly by the ovalbumin promoter. On the other hand, hIgG was not induced by the ovalbumin promoter-driven Cre in chicken embryonic fibroblast cells.</p> <p>Conclusions</p> <p>The Cre/<it>lox</it>P-based system could significantly increase ovalbumin promoter-driven production of proteins of interest, specifically in oviduct cells. This expression system could be useful for producing therapeutic mAbs at high level using transgenic chickens as bioreactors.</p

ヨウチエン デノ プレイ セラピー ノ ジッセン ケンキュウ : ヨウジ ノ ソダツ チカラ ト コソダテ シエン トシテノ コウカ

Collaboration was organized between A university and two kindergartens in A city and play therapy was performed with nine children using empty kindergarten classrooms. University teachers with a clinical psychology qualification held discussions with the parents of these children, and kindergarten teachers provided consultation to the parents and exchanged information with them. The effectiveness of play therapy was verified on the basis of a questionnaire conducted before and after play therapy implementation. The results showed that after play therapy, the children became emotionally stable, had fewer behavior problems, and showed an improvement in their social nature. Moreover, seven out of the nine parents perceived that their children had matured. The kindergarten teachers presented the same evaluation from their results. The practice records showed that the children gained confidence and pride by enjoying playing with therapists and being accepted by them, which led to them taking interest in their friends. Their growth potential was activated. In addition, changes in parent−child relationships were observed. Play therapy also supported the parents, and it was considered an effective child raising support measure

Production of Recombinant Monoclonal Antibodies in the Egg White of Gene-Targeted Transgenic Chickens

Increased commercial demand for monoclonal antibodies (mAbs) has resulted in the urgent need to establish efficient production systems. We previously developed a transgenic chicken bioreactor system that effectively produced human cytokines in egg whites using genome-edited transgenic chickens. Here, we describe the application of this system to mAb production. The genes encoding the heavy and light chains of humanized anti-HER2 mAb, linked by a 2A peptide sequence, were integrated into the chicken ovalbumin gene locus using a CRISPR/Cas9 protocol. The knock-in hens produced a fully assembled humanized mAb in their eggs. The mAb expression level in the egg white was 1.4&ndash;1.9 mg/mL, as determined by ELISA. Furthermore, the antigen binding affinity of the anti-HER2 mAb obtained was estimated to be equal to that of the therapeutic anti-HER2 mAb (trastuzumab). In addition, antigen-specific binding by the egg white mAb was demonstrated by immunofluorescence against HER2-positive and -negative cells. These results indicate that the chicken bioreactor system can efficiently produce mAbs with antigen binding capacity and can serve as an alternative production system for commercial mAbs