Library · Biology & lab methods
What Targeted Gene Overexpression got wrong, from 21 theses
Summary written by a model from the records below: Targeted gene overexpression frequently results in experimental failures, including metabolic imbalances, severe cellular toxicity, and unphysiological expression levels. Across various host systems, researchers also encounter issues where overexpressed targets fail to translate, traffic properly, or attain an active functional state. These records come from graduate theses, 2021 to 2024, each labeled with its degree where the repository states it, and each links to its thesis. They were extracted by language models reading the full text, so treat each as a lead to read, not a verdict.
Plus 7 problems the authors left open, listed at the end.
Failure modes, by theses reporting them
Select a mode to jump to its records.
Pathway imbalance and metabolic burden impairing product yields or cellular growth
Overexpressing individual pathway enzymes, regulators, or transporters often reduces target metabolite accumulation instead of improving pathway flux. In multiple engineered hosts, high expression depletes metabolic intermediates, causes severe growth burdens, and can suppress downstream enzyme activity.
Tried and failed
regulatory protein overexpression for secondary metabolite enhancement applied to bacterial biocontrol against fungal growth. Outcome: worse than baseline. Reason: overexpression impaired host growth rate without increasing antifungal efficacy over the wild-type strain
Engineering the Skin Microbiome for Drug Delivery · Georgia Tech · PhD
Tried and failed
Overexpression of native sugar transporters applied to Microbial target metabolite biosynthesis. Outcome: worse than baseline. Reason: Overexpression reduced product titers rather than enhancing substrate uptake or pathway flux
Bioproduction of D-Tagatose in Escherichia coli by Harnessing the Reverse Leloir Pathway · Harvard · Master's
Tried and failed
enzymatic overexpression to boost substrate synthesis applied to cellular protein ribosylation profiling. Outcome: worse than baseline. Reason: overexpressing the synthesis enzyme unexpectedly and almost completely suppressed downstream target enzyme activity
Targeting NAD+ metabolism in ovarian cancer · Imperial · PhD
Tried and failed
increasing transporter expression with strong promoters applied to bacterial nutrient uptake and bioproduction. Outcome: worse than baseline. Reason: overexpression caused metabolic burden and reduced uptake, biomass, and product formation
Enhancing biomolecule production through microbial engineering and advanced manufacturing · UT Austin · PhD
Tried and failed
concurrent overexpression of precursor supply pathway genes applied to heterologous metabolic pathway flux. Outcome: worse than baseline. Reason: overexpressing the citrate route decreased target polyketide biosynthesis
Developing Yarrowia lipolytica as a platform for polyketide production · UT Austin · PhD
Tried and failed
individual gene overexpression in metabolic pathway applied to terpenoid bioproduction in yeast. Outcome: worse than baseline. Reason: overexpressing specific bottleneck enzymes disrupted pathway balance and reduced target product yield
Synthetic microbial communities and multiplexed metabolic tuning by CRISPRai for enhanced bioproduction · Imperial · PhD
Severe cytotoxicity and genomic instability caused by high expression levels
High levels of target protein expression often trigger severe host cell toxicity, growth burdens, and substantial viability loss. In several experimental systems, this excessive expression suppresses reporter detection or drives counter-selection through promoter deletions and loss-of-function mutations.
Considered and rejected
Considered and rejected: Rejected using constant high-level IPTG induction for acrAB-sfgfp assays due to severe overexpression growth burdens.
Dynamics and evolution of efflux pump-mediated antibiotic resistance · Boston University · PhD
Tried and failed
plasmid-based genetic complementation via gene overexpression applied to multi-gene deletion bacterial mutants. Reason: overexpression of the target gene caused severe cellular toxicity, preventing rescue of the deletion mutant
Lytic Transglycosylases: Untangling Misconceptions in Bacterial Cell Wall Dynamics · Cornell · PhD
Tried and failed
transient plasmid co-transfection for reporter assays applied to DNA double-strand break repair reporter cells. Reason: overexpression induced widespread cytotoxicity and suppressed fluorescent reporter expression
Molecular determinants of radiosensitivity in Merkel cell carcinoma · Harvard · PhD
Tried and failed
strong constitutive overexpression of membrane transporter genes applied to bacterial secretion system refactoring. Outcome: unstable. Reason: Transporter overexpression was toxic, driving promoter deletions and loss-of-function mutations.
Developing a Broad-Host-Range Modular Microcin Secretion System to Protect Plants from Bacterial Pathogens · UT Austin
Tried and failed
direct C-terminal fluorescent protein tagging via lentivirus applied to enzyme overexpression reporter systems. Reason: cytotoxic byproduct overexpression led to poor transduction efficiency and cell viability loss
Temporal Regulation of Pro-Inflammatory Macrophage and Microglia Activation Dynamics Using Model Predictive Control · Georgia Tech · PhD
Excessive protein accumulation masking physiological regulation or creating artifactual phenotypes
Overexpression can obscure genuine biological mechanisms by masking loss-of-function phenotypes and drowning out regulatory responses. Elevated protein levels also cause non-specific substrate stimulation, unwanted cell fate transitions, or aberrant signaling correlations that diverge from endogenous behavior.
Tried and failed
exogenous mutant gene overexpression in knockout cells applied to phenotypic disease mutation modeling. Outcome: did not generalise. Reason: overexpression caused non-specific substrate stimulation rather than the selective impairment seen in endogenous mutant cells
Effect of iRhom2 Cytotail Mutations on ADAM17 Function in Vivo: Implications For Tylosis with Oesophageal Cancer · Cornell · PhD
Tried and failed
titrated transcriptional repression to normalize expression levels applied to transgenic calcium indicator overexpression toxicity. Reason: could not reduce aberrant network correlations without dropping indicator expression below usable detection levels
Optical investigation of microcircuit computations in mouse primary visual cortex · Harvard · PhD
Tried and failed
transient overexpression co-transfection assay applied to protein stabilization and regulation assays. Outcome: no signal. Reason: excessive baseline target expression masked the regulatory enhancement effect
Role of novel transmembrane protein 128 (TMEM128) in the regulation of NOX2 expression and ER Stress · Imperial · Master's
Tried and failed
Transcription factor overexpression screening applied to Stem cell pluripotency maintenance. Reason: Target factors induced premature loss of pluripotency markers and caused divergence to alternative fates
Cell fate choice and morphogenesis in stem cell models of early human development · Harvard · PhD
Considered and rejected
Considered and rejected: Evaluating receptor motif functions using high-copy 10XUAS/Gal4 overexpression lines, which masked loss-of-function phenotypes due to excessive protein accumulation
Mechanisms Of Axon Guidance Receptor Regulation And Signaling During Midline Crossing · Penn · PhD
Failure to achieve functional accumulation, translation, or active target states
Engineered overexpression constructs often fail to produce detectable target protein due to translational failure or ineffective transcriptional activation. Even when expressed, the target proteins may lack critical post-translational modifications, remain functionally inactive, or suffer from defective subcellular trafficking.
Considered and rejected
Considered and rejected: Rejected pure HSF1 genetic overexpression for inducing transcriptional activity because overexpressed HSF1 remains largely inactive
Leveraging HSF1 chemical-genetic tools to elucidate mechanisms of proteostasis · MIT · PhD
Tried and failed
CRISPR-dCas9 transcriptional activation applied to endogenous gene overexpression in germline cells. Outcome: no signal. Reason: Target protein levels showed no detectable increase upon activation
Surface behaviors in development: pattern formation in the cell cortex and nuclear envelope · Harvard · PhD
Considered and rejected
Considered and rejected: Abandoned p35S::AHP4 overexpression lines due to failure of protein translation/accumulation, prioritizing CRISPR knockout mutants instead.
Exploring the Role of ARABIDOPSIS HISTIDINE PHOSPHOTRANSFERASE 4 (AHP4) in Cytokinin Signalling · Nottingham · PhD
Considered and rejected
Considered and rejected: Abandoned doxycycline-inducible monoclonal Flp-In tBFP-RHBDL2 cell lines because stable expression produced weaker cleavage and defective surface trafficking compared to transient overexpression of tBFP-RHBDL2.
Discovery and validation of novel substrates for the rhomboid protease RHBDL2 · Oxford · PhD
Tried and failed
metabolic model guided targeted gene overexpression applied to mammalian cell line engineering. Reason: the targeted gene failed to translate into detectable protein in the engineered cell line
Utilising a genome scale metabolic model to design high-producing CHO cell lines through bi-level optimisation · Imperial · PhD
Left open by the authors
Problems the authors named and did not get to.
Left open
Compare tunable histone mimic incorporation against broad histone writer overexpression (SUV39H1, MYST1) during cell fate decision assays. Blocker: Requires molecular biology wet lab, cell lines (NIH-3T3), and plasmid constructs to perform overexpression and cell fate experiments.
The role of histone modifications in regulating nuclear rigidity and fate plasticity · Harvard · PhD
Left open
Apply and evaluate the driving transcription factor identification framework on 308 human knockout and overexpression validation experiments. Blocker: None
Conditional Multifactorial Contingency (CMC) Model and Its Applications · Virginia Tech · Doctorate
Left open
Test whether dominant negative lethality from Boi1/2 or Boi1-PH overexpression is caused by Cdc42 sequestration or negative effector signaling in yeast. Blocker: Requires wet lab yeast genetics and cell biology experiments.
CHARACTERIZING CONDITIONAL MUTANTS OF BOI1/2 IN SACCHAROMYCES CEREVISIAE · Cornell · PhD
Left open
Test whether Th2 skewing in EROS deficiency is Notch/RBPJ-dependent via lentiviral RBPJ overexpression or driven by prolonged STAT6 signaling using phospho-CyTOF. Blocker: Requires wet lab experiments with primary human CD4+ T cells, lentiviral transduction, and phospho-CyTOF apparatus
The role of EROS in T cell biology · Imperial · PhD
Left open
Perform CRISPR-Cas9 loss-of-function mutations or overexpression of candidate genes to functionally validate identified chemoresistant transcriptomic signatures. Blocker: Requires a wet-lab environment, cell cultures, and CRISPR-Cas9 experimental assays.
Clonal deconvolution of transcriptomic signatures and their spatial organisation in a mouse model of breast cancer · Cambridge · PhD
Left open
Validate Cdh1 overexpression conclusions in human embryonic stem cells using CRISPR-mediated activation instead of the DHFR-TMP stabilization system. Blocker: Requires wet lab facilities, cell culture, and CRISPR genetic engineering apparatus.
Cell cycle remodelling during human embryonic stem cell differentiation · Imperial · PhD
Left open
Pinpoint specific limiting cellular resources, such as transcription factors or ER machinery, to engineer host overexpression strains. Blocker: Requires a wet lab for mammalian cell culture, transfection, screening, and genetic engineering.
Resource-aware mammalian cell engineering · Imperial · PhD
Who has worked on this
Authors of the public theses cited on this page, most-cited first.
- Roya Huang · Harvard · PhD · 2024
Cell fate choice and morphogenesis in stem cell models of early human development - Jonathan A Jackson · Harvard · PhD · 2024
Surface behaviors in development: pattern formation in the cell cortex and nuclear envelope - Michael Gruet · Imperial · PhD · 2023
Targeting NAD+ metabolism in ovarian cancer - Veronica Montgomery · Georgia Tech · PhD · 2023
Engineering the Skin Microbiome for Drug Delivery - Roberto Di Blasi · Imperial · PhD · 2023
Resource-aware mammalian cell engineering - Ariana Rabinowitsch · Cornell · PhD · 2023
Effect of iRhom2 Cytotail Mutations on ADAM17 Function in Vivo: Implications For Tylosis with Oesophageal Cancer - Joseph Padgett · Imperial · PhD · 2023
Cell cycle remodelling during human embryonic stem cell differentiation - Christopher Toomey · Harvard · Master's · 2023
Bioproduction of D-Tagatose in Escherichia coli by Harnessing the Reverse Leloir Pathway - Elina Chrysanthou · Nottingham · PhD · 2023
Exploring the Role of ARABIDOPSIS HISTIDINE PHOSPHOTRANSFERASE 4 (AHP4) in Cytokinin Signalling - L Zarcone · Oxford · PhD · 2023
Discovery and validation of novel substrates for the rhomboid protease RHBDL2 - Zuolin Cheng · Virginia Tech · Doctorate · 2023
Conditional Multifactorial Contingency (CMC) Model and Its Applications - Amalia Driller-Colangelo · Harvard · PhD · 2022
The role of histone modifications in regulating nuclear rigidity and fate plasticity
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