Choice of selectable marker affects recombinant protein expression in cells and exosomes.
Choice of selectable marker affects recombinant protein expression in cells and exosomes.
复制标题
选择标记的选择会影响细胞和外泌体中的重组蛋白表达。
DOI：
10.1016/j.jbc.2021.100838
复制
发表时间：
2021-07
期刊：
影响因子：
--
通讯作者：
Gould SJ
中科院分区：
文献类型：
--
作者：
Guo C;Fordjour FK;Tsai SJ;Morrell JC;Gould SJ
转基因哺乳动物细胞用于许多研究、制药、工业和临床目的，并且通常使用显性选择标记来选择转基因细胞系。使用 HEK293 细胞，我们在此表明，选择性标记基因的选择对重组蛋白表达水平和重组蛋白表达的细胞间变异性均具有显着影响。具体来说，我们观察到用 NeoR 或 BsdR 选择标记生成并分别在抗生素 G418 或杀稻瘟菌素中选择的细胞系显示出最低水平的重组蛋白表达以及最大的细胞间转基因表达变异性。相比之下，使用 BleoR 标记生成并在博莱霉素中选择的细胞系产生的细胞系表达最高水平的连接重组蛋白，比使用 NeoR 或 BsdR 标记选择的细胞系高约 10 倍，并且重组蛋白表达的细胞间变异性最低。在使用基于 PuroR 或 HygR 的载体生成的细胞中观察到中等但仍然高水平的表达，并且分别在嘌呤霉素或潮霉素中进行选择。在非洲绿猴细胞系 COS7 中也观察到类似的结果。这些数据表明，选择标记和抗生素的每种组合都建立了一个阈值，低于该阈值则没有细胞能够存活，并且这些阈值在不同的选择标记之间显着变化。此外，我们表明选择标记的选择也会影响细胞源性外泌体中的重组蛋白表达，这与外泌体蛋白出芽是随机而非决定性过程的假设一致。
Transgenic mammalian cells are used for numerous research, pharmaceutical, industrial, and clinical purposes, and dominant selectable markers are often used to enable the selection of transgenic cell lines. Using HEK293 cells, we show here that the choice of selectable marker gene has a significant impact on both the level of recombinant protein expression and the cell-to-cell variability in recombinant protein expression. Specifically, we observed that cell lines generated with the NeoR or BsdR selectable markers and selected in the antibiotics G418 or blasticidin, respectively, displayed the lowest level of recombinant protein expression as well as the greatest cell-to-cell variability in transgene expression. In contrast, cell lines generated with the BleoR marker and selected in zeocin yielded cell lines that expressed the highest levels of linked recombinant protein, approximately 10-fold higher than those selected using the NeoR or BsdR markers, as well as the lowest cell-to-cell variability in recombinant protein expression. Intermediate yet still-high levels of expression were observed in cells generated with the PuroR- or HygR-based vectors and that were selected in puromycin or hygromycin, respectively. Similar results were observed in the African green monkey cell line COS7. These data indicate that each combination of selectable marker and antibiotic establishes a threshold below which no cell can survive and that these thresholds vary significantly between different selectable markers. Moreover, we show that choice of selectable marker also affects recombinant protein expression in cell-derived exosomes, consistent with the hypothesis that exosome protein budding is a stochastic rather than determinative process.
登录
查看更多内容
影响因子：
64.5
作者：
Hsu PD;Lander ES;Zhang F
通讯作者：
Zhang F
影响因子：
16
作者：
Bachurski, Daniel;Schuldner, Maximiliane;von Strandmann, Elke Pogge
通讯作者：
von Strandmann, Elke Pogge
影响因子：
5.1
作者：
Chen, ZY;He, CY;Kay, MA
通讯作者：
Kay, MA
DOI：
10.1073/pnas.78.4.2072
发表时间：
1981-01-01
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
--
作者：
MULLIGAN, RC;BERG, P
通讯作者：
BERG, P
影响因子：
14.9
作者：
KASTER, KR;BURGETT, SG;INGOLIA, TD
通讯作者：
INGOLIA, TD