Control of Multigene Expression Stoichiometry in Mammalian Cells Using Synthetic Promoters.
Control of Multigene Expression Stoichiometry in Mammalian Cells Using Synthetic Promoters.
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DOI：
10.1021/acssynbio.0c00643
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发表时间：
2021-05-21
影响因子：
4.7
通讯作者：
James DC
中科院分区：
文献类型：
--
作者：
Patel YD;Brown AJ;Zhu J;Rosignoli G;Gibson SJ;Hatton D;James DC
为了成功地改造哺乳动物细胞以达到预期目的，需要多个重组基因以特定的最佳比例共表达。在这项研究中，我们假设合成的转录活性不同的启动子可以在可预测的相对化学计量中用于创建单个多基因表达载体，共表达重组基因。建立了一个包含27个多基因构建体的文库，包括三个固定序列的离散荧光报告基因转录单位，每个单位在每种可能的组合中分别由相对低、中或高转录强度的合成启动子控制。采用绝对定量qRT-PCR法检测CHO细胞中各报告基因的表达。合成的启动子在多基因载体环境中确实发挥了设计的作用；然而，与预测的启动子介导的转录活性存在显著差异。首先，相对于相同的报告基因在独立的单基因载体上在相同的基因拷贝上的共表达，多基因载体中所有三种基因的表达在不同程度上受到抑制。其次，基因位置效应在所有构建体中都很明显，其中位置2和3的报告基因的表达通常相对于位置1减少。最后，在考虑了一般抑制后，局部多基因载体格式中的合成启动子转录活性偏离了预期。综上所述，我们的数据表明，哺乳动物合成启动子可以在可预测的相对化学计量中用于载体介导多个基因的表达。然而，功能性能的实证验证是必要的先决条件，因为向量和启动子设计特征会显著影响性能。
To successfully engineer mammalian cells for a desired purpose, multiple recombinant genes are required to be coexpressed at a specific and optimal ratio. In this study, we hypothesized that synthetic promoters varying in transcriptional activity could be used to create single multigene expression vectors coexpressing recombinant genes at a predictable relative stoichiometry. A library of 27 multigene constructs was created comprising three discrete fluorescent reporter gene transcriptional units in fixed series, each under the control of either a relatively low, medium, or high transcriptional strength synthetic promoter in every possible combination. Expression of each reporter gene was determined by absolute quantitation qRT-PCR in CHO cells. The synthetic promoters did generally function as designed within a multigene vector context; however, significant divergences from predicted promoter-mediated transcriptional activity were observed. First, expression of all three genes within a multigene vector was repressed at varying levels relative to coexpression of identical reporter genes on separate single gene vectors at equivalent gene copies. Second, gene positional effects were evident across all constructs where expression of the reporter genes in positions 2 and 3 was generally reduced relative to position 1. Finally, after accounting for general repression, synthetic promoter transcriptional activity within a local multigene vector format deviated from that expected. Taken together, our data reveal that mammalian synthetic promoters can be employed in vectors to mediate expression of multiple genes at predictable relative stoichiometries. However, empirical validation of functional performance is a necessary prerequisite, as vector and promoter design features can significantly impact performance.
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