Emerging synthetic biology tools for engineering mammalian cell systems and expediting cell line development

Lanza, Amanda M.; Cheng, J.; Alper, Hal S.

doi:10.1016/j.coche.2012.09.005

Cited by 4 publications

(5 citation statements)

References 76 publications

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“…The effectiveness of selection directly influences the quality of the selected pool and the resulting single cell clones. As the demand for metabolic engineering of mammalian cell systems increases, and thereby precise gene expression, it is important to consider the influence synthetic control elements like selection markers have on recombinant gene expression [21]. Several selection markers (and corresponding agents) are widely available for mammalian cells; however, their efficacy has not been compared in a single study.…”

Section: Introductionmentioning

confidence: 99%

Evaluating the influence of selection markers on obtaining selected pools and stable cell lines in human cells

Lanza

Kim

Alper

2013

Biotechnology Journal

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Selection markers are common genetic elements used in recombinant cell line development. While several selection systems exist for use in mammalian cell lines, no previous study has comprehensively evaluated their performance in the isolation of recombinant populations and cell lines. Here we examine four antibiotics, hygromycin B, neomycin, puromycin, and Zeocin™, and their corresponding selector genes, using a green fluorescent protein (GFP) as a reporter in two model cell lines, HT1080 and HEK293. We identify Zeocin™ as the best selection agent for cell line development in human cells. In comparison to the other selection systems, Zeocin™ is able to identify populations with higher fluorescence levels, which in turn leads to the isolation of better clonal populations and less false positives. Furthermore, Zeocin™-resistant populations exhibit better transgene stability in the absence of selection pressure compared to other selection agents. All isolated Zeocin™-resistant clones, regardless of cell type, exhibited GFP expression. By comparison, only 79% of hygromycin B-resistant, 47% of neomycin-resistant, and 14% of puromycin-resistant clones expressed GFP. Based on these results, we rank Zeocin™ > hygromycin B ∼ puromycin > neomycin for cell line development in human cells. Furthermore, this study demonstrates that selection marker choice does indeed impact cell line development.

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Section: Introductionmentioning

confidence: 99%

Evaluating the influence of selection markers on obtaining selected pools and stable cell lines in human cells

Lanza

Kim

Alper

2013

Biotechnology Journal

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“…In contrast to the large and growing variety of transcription factors and corresponding REs for mammalian synthetic biology, relatively few options exist in the choice of core promoters . The majority of mammalian inducible promoters reported thus far are constructed from a small selection of core promoters, most prominently the minimal cytomegalovirus (minCMV) promoter. ,− Although these tried-and-true promoters have enabled the demonstration of diverse functional outputs, they do not necessarily exhibit optimal regulatory behavior.…”

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confidence: 99%

Quantitative Analyses of Core Promoters Enable Precise Engineering of Regulated Gene Expression in Mammalian Cells

et al. 2016

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Inducible transcription systems play a crucial role in a wide array of synthetic biology circuits. However, the majority of inducible promoters are constructed from a limited set of tried-and-true promoter parts, which are susceptible to common shortcomings such as high basal expression levels (i.e., leakiness). To expand the toolbox for regulated mammalian gene expression and facilitate the construction of mammalian genetic circuits with precise functionality, we quantitatively characterized a panel of eight core promoters, including sequences with mammalian, viral, and synthetic origins. We demonstrate that this selection of core promoters can provide a wide range of basal gene expression levels and achieve a gradient of fold-inductions spanning two orders of magnitude. Furthermore, commonly used parts such as minimal CMV and minimal SV40 promoters were shown to achieve robust gene expression upon induction, but also suffer from high levels of leakiness. In contrast, a synthetic promoter, YB_TATA, was shown to combine low basal expression with high transcription rate in the induced state to achieve significantly higher fold-induction ratios compared to all other promoters tested. These behaviors remain consistent when the promoters are coupled to different genetic outputs and different response elements, as well as across different host-cell types and DNA copy numbers. We apply this quantitative understanding of core promoter properties to the successful engineering of human T cells that respond to antigen stimulation via chimeric antigen receptor signaling specifically under hypoxic environments. Results presented in this study can facilitate the design and calibration of future mammalian synthetic biology systems capable of precisely programmed functionality.

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“…These diverse, synthetic sequences comprise a suite of non viral-derived sequences, potentially reducing their susceptibility of epigenetic silencing and increasing long-term stability. By adjusting the TFBS composition and arrangement, a set of de novo promoters can be developed to properly tune the protein intermediates in a metabolic pathway of mammalian hosts as one of the tools governing transgene expression . Moreover, these synthetic promoters had an overall reduced sequence footprint compared to reference promoters, thus increasing the overall utility for applications such as (heterologous) metabolic pathway designs and gene delivery vector designs, akin to efforts in reducing the regulatory sequence footprint in a conventional metabolic engineering host .…”

Section: Discussionmentioning

confidence: 99%

“…By adjusting the TFBS composition and arrangement, a set of de novo promoters can be developed to properly tune the protein intermediates in a metabolic pathway of mammalian hosts as one of the tools governing transgene expression. 47 Moreover, these synthetic promoters had an overall reduced sequence footprint compared to reference promoters, thus increasing the overall utility for applications such as (heterologous) metabolic pathway designs 6 and gene delivery vector designs, 48 akin to efforts in reducing the regulatory sequence footprint in a conventional metabolic engineering host. 49 It is possible to expand the combinatorial space in this work to enable many permutations of synthetic randomized concatenations of a particular set of TFBSs to be constructed and evaluated.…”

Section: ■ Conclusionmentioning

confidence: 99%

Transcriptomics-Guided Design of Synthetic Promoters for a Mammalian System

Cheng

Alper

2016

ACS Synth. Biol.

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Despite recent advances in improving titers for therapeutic proteins such as antibodies to the 10 g/L scale, these high yields can only be achieved in select mammalian hosts. Regardless of the host or product, strong promoters are required to obtain high levels of transgene expression. However, the promoters employed to drive this expression are rather limited in variety and are usually either viral-derived or screened empirically during vector design. To begin to move away from viral parts, we employed a more systematic approach to identify and design new synthetic promoters using endogenous elements. To do so, we established a workflow to design these elements by (1) analyzing the transcriptomics profile of a specific cell line under a desired, representative cell culture condition, (2) identifying key genetic motifs using bioinformatics that can be used to rationally construct synthetic promoters, (3) building synthetic promoters using conventional DNA synthesis and molecular biology techniques, and (4) evaluating the performance of these synthetic promoters using model proteins. The resulting promoters perform comparably to the hCMV IE promoter variants tested, but with endogenous components. During this design-build-test cycle we also investigated the underlying design rules for transcription factor binding site arrangement in synthetic promoters. Overall, this approach of using an "omics-guided" workflow for designing synthetic promoters facilitates the construction of high expression vectors for immediate use in current production hosts.

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Emerging synthetic biology tools for engineering mammalian cell systems and expediting cell line development

Cited by 4 publications

References 76 publications

Evaluating the influence of selection markers on obtaining selected pools and stable cell lines in human cells

Evaluating the influence of selection markers on obtaining selected pools and stable cell lines in human cells

Quantitative Analyses of Core Promoters Enable Precise Engineering of Regulated Gene Expression in Mammalian Cells

Transcriptomics-Guided Design of Synthetic Promoters for a Mammalian System

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