Deciphering the Design Rules of Toehold-Gated sgRNA for Conditional Activation of Gene Expression and Protein Degradation in Mammalian Cells

2022 ◽  
Author(s):  
Victoria M. Hunt ◽  
Wilfred Chen
Keyword(s):  
Gene Expression ◽  
Protein Degradation ◽  
Mammalian Cells ◽  
Design Rules

scholarly journals High-performance chemical- and light-inducible recombinases in mammalian cells and mice

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Benjamin H. Weinberg ◽  
Jang Hwan Cho ◽  
Yash Agarwal ◽  
N. T. Hang Pham ◽  
Leidy D. Caraballo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
High Performance ◽  
Genome Engineering ◽  
Genetic Circuits ◽  
Control Of Gene Expression ◽  
Expression Control ◽  
Gene Expression Control ◽  
High Performance Systems ◽  
Spatiotemporal Control

Abstract Site-specific DNA recombinases are important genome engineering tools. Chemical- and light-inducible recombinases, in particular, enable spatiotemporal control of gene expression. However, inducible recombinases are scarce due to the challenge of engineering high performance systems, thus constraining the sophistication of genetic circuits and animal models that can be created. Here we present a library of >20 orthogonal inducible split recombinases that can be activated by small molecules, light and temperature in mammalian cells and mice. Furthermore, we engineer inducible split Cre systems with better performance than existing systems. Using our orthogonal inducible recombinases, we create a genetic switchboard that can independently regulate the expression of 3 different cytokines in the same cell, a tripartite inducible Flp, and a 4-input AND gate. We quantitatively characterize the inducible recombinases for benchmarking their performances, including computation of distinguishability of outputs. This library expands capabilities for multiplexed mammalian gene expression control.

scholarly journals Integrated Genomic and Proteomic Analyses of Gene Expression in Mammalian Cells

2004 ◽  
Vol 3 (10) ◽  
pp. 960-969 ◽  
Author(s):  
Qiang Tian ◽  
Serguei B. Stepaniants ◽  
Mao Mao ◽  
Lee Weng ◽  
Megan C. Feetham ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
Proteomic Analyses ◽  
Expression In Mammalian Cells

scholarly journals Characterization, modelling and mitigation of gene expression burden in mammalian cells

2019 ◽  
Author(s):  
T Frei ◽  
F Cella ◽  
F Tedeschi ◽  
J Gutierrez ◽  
GB Stan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
Genome Engineering ◽  
Host Cells ◽  
Genetic Circuits ◽  
Circuit Performance ◽  
Resource Requirements ◽  
Synthetic Circuit ◽  
Resource Aware

AbstractDespite recent advances in genome engineering, the design of genetic circuits in mammalian cells is still painstakingly slow and fraught with inexplicable failures. Here we demonstrate that competition for limited transcriptional and translational resources dynamically couples otherwise independent co-expressed exogenous genes, leading to diminished performance and contributing to the divergence between intended and actual function. We also show that the expression of endogenous genes is likewise impacted when genetic payloads are expressed in the host cells. Guided by a resource-aware mathematical model and our experimental finding that post-transcriptional regulators have a large capacity for resource redistribution, we identify and engineer natural and synthetic miRNA-based incoherent feedforward loop (iFFL) circuits that mitigate gene expression burden. The implementation of these circuits features the novel use of endogenous miRNAs as integral components of the engineered iFFL device, a versatile hybrid design that allows burden mitigation to be achieved across different cell-lines with minimal resource requirements. This study establishes the foundations for context-aware prediction and improvement of in vivo synthetic circuit performance, paving the way towards more rational synthetic construct design in mammalian cells.

scholarly journals Probability in transcriptional regulation and its implications for leukocyte differentiation and inducible gene expression

Blood ◽  
2000 ◽  
Vol 96 (7) ◽  
pp. 2323-2328 ◽  
Author(s):  
David A. Hume
Keyword(s):  
Gene Expression ◽  
Transcriptional Regulation ◽  
Mammalian Cells ◽  
Cell Lineage ◽  
Monoallelic Expression ◽  
Stem Cell Lineage ◽  
The Stability ◽  
Stochastic Phenomena ◽  
Inducible Genes ◽  
Probabilistic Nature

The phenotype of individual hematopoietic cells, like all other differentiated mammalian cells, is determined by selective transcription of a subset of the genes encoded within the genome. This overview summarizes the recent evidence that transcriptional regulation at the level of individual cells is best described in terms of the regulation of the probability of transcription rather than the rate. In this model, heterogeneous gene expression among populations of cells arises by chance, and the degree of heterogeneity is a function of the stability of the mRNA and protein products of individual genes. The probabilistic nature of transcriptional regulation provides one explanation for stochastic phenomena, such as stem cell lineage commitment, and monoallelic expression of inducible genes, such as lymphokines and cytokines.

The Leeuwenhoek Lecture, 1978 Bacteria as proper subjects for cancer research

1980 ◽  
Vol 208 (1171) ◽  
pp. 121-133 ◽  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Cancer Research ◽  
Mammalian Cells ◽  
Cell Renewal ◽  
Epigenetic Alterations ◽  
Cell Lineages ◽  
Abnormal Cells ◽  
Kinetics Of

Cancers are clones of abnormal cells, arising presumably as the result of mutational or epigenetic alterations of gene expression. The kinetics of appearance of spontaneous cancers in populations of multiplying cells (i. e. the relation between age and cancer incidence) will therefore depend, among other things, on how these populations are organized and, in general, on the kinetics of the response of cells to prolonged mutagenesis. The organization of cell renewal in epithelia (i. e. the arrangement of cell lineages) is still rather obscure; in particular, it is not known to what extent the properties and organization of the stem cells tend to protect them from accumulating mutations. We have tried to mimic the arrangement of epithelia by attaching multiplying bacteria to filters. Study of mutagenesis in long-term cultures of such anchored bacteria has led to the discovery of some additional pathways for DNA repair which also appear to operate in mammalian cells.

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