中国科学院昆明动物研究所机构知识库

家蚕（Bombyxmori）是鳞翅目昆虫的模式生物，起源于中国的古野桑蚕，人工驯化历史已经有5000多年。由其形成的产业曾为中国传统政治、经济和文化作出了卓越的贡献。但是，随着人造聚合纤维的发明以及我国经济结构转变和产业转型升级，我国的蚕桑丝绸产业面临了前所未有的挑战。随着越来越多物种的基因组数据被公布，在更为广泛的物种中开展功能基因组学研究已经越来越迫切。家蚕完整基因组的解析为家蚕的后基因组时代的研究打下了良好的基础，蚕丝产业已经步入了必须依靠重大技术突破和思路创新的新阶段。近年来，基因组编辑技术，如锌指核酸酶技术(ZFNs)和TALE核酸酶技术(TALENs)，尤其是最新发展的CRISPR/Cas9基因编辑技术，带来了功能基因组研究的革新，并且基于CRISPR/Cas9技术的基因改造工具的发展速度可谓一日千里。尤其是可在一个基因的两个副本产生突变的MCR(Mutagenic Chain Reaction)技术。这类高效基因编辑技术，目前已在模式生物果蝇和非传统模式生物蚊子研究过程中取得了重要突破。本论文首次以家蚕和家蚕细胞为对象，以MCR技术为基础，初步探讨了开发家蚕转基因操作新工具的可能性。丝胶蛋白是蚕丝表面的一类可溶蛋白，在细胞培养、化妆品、保健食品及生物材料等领域都具有重要的应用价值。随着细胞培养技术已被广泛应用于现代分子生物学和生物产业，其培养基组分的优化和替代需求日趋紧迫。血清作为目前大部分哺乳动物细胞培养的必须成分也有着其本身致命的缺陷，如支原体、病毒和朊病毒等微生物污染。而家蚕丝胶蛋白作为一种新兴的血清替代产品其应用前景广泛，但缺乏统一系统的评估，其应用的普遍性还未知。为此，我们首次结合细胞以及染色体形态学、细胞生理学和细胞转录组分析对丝胶蛋白替代血清培养细胞进行了综合评估。本论文以家蚕、家蚕细胞以及丝胶蛋白为对象，初步探讨了开发在家蚕遗传操作的G0代能产生纯合基因型的精准转基因遗传工具的可能性；同时较为系统地评估了丝胶蛋白替代细胞培养基中的血清后细胞的生长状态、代谢活性、染色体倍性以及基因转录水平的表达丰度等。结果如下：1. 根据MCR转基因策略并结合无缝连接技术，将Cas9基因与识别切割靶位点的gRNAs以及标记基因连同切割靶位点附近的同源臂一起，成功构建MCR转基因载体系统。通过细胞转染、基因枪导入和显微注射将该MCR转基因载体导入家蚕DZNU-Bm-12细胞系或者家蚕卵的实验结果表明MCR转基因载体在细胞或卵中正确响应，标记基因表达活性正常。2. 通过显微注射家蚕卵导入的MCR转基因载体(pCas9-EGFP质粒)，成功地利用CRISPR/Cas9介导，将含有Cas9的基因表达盒精准替换了家蚕BmFIBH基因，并且在G0代个体中检测到替换后的纯合基因型。该精准转基因体系为功能基因组研究开拓了崭新的技术。该体系显著提高了定点转基因操作的效率并且缩短了产生纯合子的时间。我们目前正在尝试利用该技术导入富含重复序列的天蚕丝素基因到家蚕基因组中改良家蚕丝质。3. 首次结合细胞以及染色体形态学、细胞生理学和细胞转录组分析对三类广泛使用的哺乳动物细胞系(CHO细胞、MARC-145细胞和HeLa细胞)在丝胶蛋白替代血清的培养环境下的生长状态、细胞活性、染色体倍性以及基因表达丰度进行评估。结果表明，丝胶替代血清维持哺乳动物细胞的生长和繁殖的效果与含血清的细胞培养基相当或更好。本文不仅为丝胶蛋白替代血清培养细胞提供了确凿的实验证据，还有助于对细胞培养和丝绸产业可持续发展的理解。

The domesticated silkworm (Bombyx mori) is model species of the lepidopteran, which was derived from wild silkworm (Bombyx mandarina) in China and has more than 5,000 years’s history. It made outstanding contributions by sericulture in the politics, economy and culture in China. However, with the development of man-made artificial polymer fiber products, economic structure and industrial transformation in China, the sericulture is faced with unprecedented challenges. With more and more reference genomes available, it become urgent to carry out functional genomics research in a wider range of species. The complete genome seqence of Bombyx makes good foundations of its post-genomic era study. Silk industry has entered a new stage of development, which must rely on innovative ideas and major technological breakthroughs.In recent years, genome editing techniques like ZFNs, TALENs, especially, the recent developed CRISPR-Cas9 has bring the research innovation of functional genomics. Moreover, the improvements and derivative products based on CRISPR-Cas9 have come along by leaps and bounds, such as MCR. MCR is one of its derivative product, which is based on the CRISPR/Cas9 genome-editing system for generating autocatalytic mutations, to produce homozygous loss-of-function mutations.Sericin is a class of soluble protein on the surface of silk, which has significant application value the fields of cell culture, cosmetics, health food and biological materials. As the cell culture technology has been widely used in modern cell biology and biotechnology industry, the optimization and replacement of medium components becoming increasingly urgent. Serum, the most important supplement in the traditional medium for the culture of mammalian cells, has many fatal flaws such as the contamination by microbes like Mycoplasma, viruses, and prions. The replacement of serum by sericin, has a wider application prospects, but it lacks of a uniform and systematical assessment and its universality application is currently unknown. We have conducted a comprehensive evaluation of sericin replacement serum in cell culture based on the morphological, physiological, and transcriptomic features for the first time.We studied the silkworm, the silkworm cell, and sericin, and preliminarily discussed the possibility of the development of precision transgenic genetic manipulation tool, which can produce homozygous genotype in G0 generation of silkworm. At the same time, we compared the cellular growth status, metabolic activity, chromosome ploidy and gene expression abundance between cells cultured in sericin-substituted medium and those cultured in conventional FBS-containing medium. The results are as follows:1. We have successfully constructed MCR plasmids, which contain Cas9, gRNAs, homologous arms and marker genes based on MCR transgenic strategy and In-Fusion assembly. The results of cell transfection, DNA particle bombardment and microinjection all shown the correct activation of plasmid activity and efficient expression of the marker genes in the DZNU-Bm-12 cells or eggs.2. We used the novel plasmid vector (pCas9-EGFP) to achieve the accurate replacement of BmFIBH in silkworm by a Cas9-based gene expression cassette. And, the homozygous genotype was detected in the G0 generation. The precise transgenic system has opened up a new avenue of functional genomics research. Because it can significantly increases the efficiency of precise transgenes and shortens the time to produce homozygotes. Transgenic strategies based on MCR may be applied to the improvement over traditional silk. Currently, we are attempting to use this technique to improve quality of silk products by knock-in Antheraea yamamai FIBH, which is rich in repetitive sequences, into the silkworm genome.3. Here, we compared cellular growth status, cell metabolic activity, chromosome ploidy and gene expression abundance between the three most commonly used cells (CHO, MARC-145 and HeLa) cultured in sericin-substituted medium and those cultured in conventional FBS-containing medium based on the morphological, physiological, and transcriptomic features. The results showed that the former performed as well as or even better than the latter in terms of all cellular features mentioned above. Our fndings not only provide conclusive evidence for the application of sericin as a replacement for FBS, but will also assist in future understanding of sustainable development in both the cell culture and silk industries.