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在使用生物工程哺乳动物转基因酶的中国仓鼠卵巢细胞中进行大型转基因整合的表征

Nicolas Marx¹, Anna-Maria Otte¹, Klaus Leitner¹

¹Department of Biotechnology, BOKU University of Natural Resources and Life Sciences, Vienna, Austria.

Biotechnology progress

|January 23, 2025

概括

一种新的生物工程哺乳动物转化酶系统 (bMLT) 能够有效和稳定地将基因集成到 CHO 细胞中,用于生产 GFP 和 trastuzumab. 这种方法可以在没有选择性压力的情况下加速高产细胞系的发展.

科学领域:

生物技术是生物技术.
分子生物学分子生物学
细胞工程细胞工程

背景情况:

哺乳动物的转位子系统对于稳定的基因整合到宿主基因组至关重要.
像CHO细胞这样的细胞系中高效和稳定的基因表达对于生物制药生产至关重要.
Myotis lucifugus的转位子为生物工程新型整合系统提供了潜力.

研究的目的:

评估由Myotis lucifugus (bMLT) 衍生的一种生物工程哺乳动物转化酶系统在中国汉姆斯特卵巢 (CHO) 细胞中基因整合的疗效.
评估bMLT.介导的基因表达 (GFP和trastuzumab) 的稳定性和效率.
调查bMLT集成站点对蜂生产力的影响.

主要方法:

用GFP记者和bMLT (DNA或mRNA) 感染CHO-K1细胞,在17周内监测表达,没有选择.
下一代测序用于分析集成站点和效率.
用trastuzumab和bMLT感染CHO-GS-/-细胞,然后对分泌细胞进行选择和分析.

主要成果:

与对照组不同,bMLT实现了高达60%的稳定GFP集成效率,维持了17周的表达.
每个细胞的最高GFP表达被观察到与较低的bMLT度.
通过bMLT介导的trastuzumab集成导致40%-55%的分泌细胞,明显高于随机集成 (0.3%-0.5%).
85%的集成序列被正确集成,其位置影响表达水平.

结论:

bMLT系统提供了一种稳定的基因整合和CHO细胞高表达的高效方法.
这种方法有助于快速生成高产细胞系,并评估基因工程策略.
基因组整合部位的选择是影响工程细胞系生产率的关键因素.

关键词:

在CHO细胞中.基因表达分析基因表达分析基因整合基因整合转换效率效率是指转换效率是指转换效率.转换酶是一种转换酶.这就是特拉斯图祖马布 (trastuzumab).

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To learn more about the function of a gene, researchers can observe what happens when the gene is inactivated or “knocked out,” by creating genetically engineered knockout animals. Knockout mice have been particularly useful as models for human diseases such as cancer, Parkinson’s disease, and diabetes.