Marx Nicolas, Otte Anna-Maria, Leitner Klaus, Sitepu Rehmadanta, Berger Thomas, Schäpertöns Veronika, Huber Christian G, Zhu Quan, Nema Sandeep, Higgins Joseph J, Borth Nicole
Department of Biotechnology, BOKU University of Natural Resources and Life Sciences, Vienna, Austria.
Bioanalytical Research Labs, Paris-Lodron University Salzburg, Salzburg, Austria.
Biotechnol Prog. 2025 May-Jun;41(3):e3524. doi: 10.1002/btpr.3524. Epub 2025 Jan 23.
We present the first use of a bioengineered mammalian transposase system derived from Myotis lucifugus (bMLT) for integration of expression vectors into the CHO genome, focusing on GFP and trastuzumab production. Initially, CHO-K1 cells are transfected with a GFP reporter and varying amounts of bMLT DNA or mRNA. GFP expression is monitored over 17 weeks without selective pressure. Transfection efficiency shows around 90% GFP-positive cells, but in control cultures GFP expression disappears after 10 days. In contrast, bMLT-treated cultures maintain stable GFP expression, with a dose-dependent integration efficiency of up to 60%. The highest GFP expression per cell is observed with lower bMLT amounts. Next-generation sequencing analysis reveals multiple integration sites, with 85% correctly integrated sequences. Next, CHO-GS cells are transfected with trastuzumab and bMLT DNA or mRNA. Cells are selected in glutamine-free medium with varying methionine sulfoximine (MSX) concentrations. Recovery is faster without MSX, and no difference is observed between bMLT DNA and mRNA transfections. bMLT-treated cultures show a higher percentage of trastuzumab-secreting cells (40%-55%) compared with random integration (0.3%-0.5%). The absence of insulators in the trastuzumab plasmid likely affects selection behavior, as integration in heterochromatic regions results in gene repression. Overall, bMLT-mediated integration proves efficient, generating stable cell pools with high expression profiles without selective pressure. The integration sites' genomic location significantly impacts productivity, with favorable regions supporting higher expression. This method shows promise for the rapid and efficient generation of high-producing cell lines and for rapid evaluation of long-term effects of different cell engineering approaches.
我们展示了首次使用源自食虫蝙蝠（Myotis lucifugus）的生物工程化哺乳动物转座酶系统（bMLT）将表达载体整合到CHO基因组中，重点是绿色荧光蛋白（GFP）和曲妥珠单抗的生产。最初，用GFP报告基因以及不同量的bMLT DNA或mRNA转染CHO-K1细胞。在无选择压力的情况下监测GFP表达17周。转染效率显示约90%的GFP阳性细胞，但在对照培养物中，GFP表达在10天后消失。相比之下，经bMLT处理的培养物保持稳定的GFP表达，整合效率高达60%，呈剂量依赖性。在较低bMLT量时观察到每个细胞的GFP表达最高。下一代测序分析揭示了多个整合位点，85%的序列正确整合。接下来，用曲妥珠单抗以及bMLT DNA或mRNA转染CHO-GS细胞。在含有不同浓度甲硫氨酸亚砜亚胺（MSX）的无谷氨酰胺培养基中选择细胞。在没有MSX的情况下恢复更快，并且在bMLT DNA和mRNA转染之间未观察到差异。与随机整合（0.3%-0.5%）相比，经bMLT处理的培养物显示出更高比例的分泌曲妥珠单抗的细胞（40%-55%）。曲妥珠单抗质粒中缺乏绝缘子可能会影响选择行为，因为在异染色质区域的整合会导致基因抑制。总体而言，bMLT介导的整合证明是有效的，在无选择压力的情况下产生具有高表达谱的稳定细胞库。整合位点的基因组位置对生产力有显著影响，有利区域支持更高的表达。这种方法对于快速高效地产生高产细胞系以及快速评估不同细胞工程方法的长期效果显示出前景。
