525. Directed Molecular Evolution of Transcription Factors in Mammalian Cells for Enhanced Directed Cell Differentiation

In vivo imaging of model organisms is heavily reliant on fluorescent proteins with high intracellular brightness. Here we describe a practical method for rapid optimization of fluorescent proteins via directed molecular evolution in cultured mammalian cells. Using this method, we were able to perform screening of large gene libraries containing up to 2x107 independent random genes of fluorescent proteins expressed in HEK cells completing one iteration directed evolution in a course of ~8 days. We employed this approach to develop a set of green and near-infrared fluorescent proteins with enhanced intracellular brightness. The developed near-infrared fluorescent proteins demonstrated high performance for fluorescent labeling of neurons in culture and in vivo in model organisms such as C.elegans, Drosophila, zebrafish, and mice. Spectral properties of the optimized near-infrared fluorescent proteins enabled crosstalk-free multicolor imaging in combination with common green and red fluorescent proteins, as well as dual-color near-infrared fluorescence imaging. The described method has a great potential to be adopted by protein engineers due to its simplicity and practicality. We also believe that the new enhanced fluorescent proteins will find wide application for in vivo multicolor imaging of small model organisms.

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Rapid Directed Molecular Evolution of Fluorescent Proteins in Mammalian Cells

Protein Science ◽

10.1002/pro.4261 ◽

2021 ◽

Author(s):

Siranush Babakhanova ◽

Erica E. Jung ◽

Kazuhiko Namikawa ◽

Hanbin Zhang ◽

Yangdong Wang ◽

...

Keyword(s):

Molecular Evolution ◽

Mammalian Cells ◽

Fluorescent Proteins ◽

Directed Molecular Evolution

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Rapid Directed Molecular Evolution of Fluorescent Proteins in Mammalian Cells

10.21203/rs.3.rs-776185/v1 ◽

2021 ◽

Author(s):

Siranush Babakhanova ◽

Erica Jung ◽

Kazuhiko Namikawa ◽

Hanbin Zhang ◽

Yangdong Wang ◽

...

Keyword(s):

Molecular Evolution ◽

Mammalian Cells ◽

High Performance ◽

Near Infrared ◽

Fluorescent Proteins ◽

Practical Method ◽

Fluorescent Labeling ◽

Model Organisms ◽

Directed Molecular Evolution

Abstract In vivo imaging of model organisms is heavily reliant on fluorescent proteins with high intracellular brightness. Here we describe a practical method for rapid optimization of fluorescent proteins via directed molecular evolution in cultured mammalian cells. Using this method, we were able to perform screening of large gene libraries containing up to 2·107 independent random genes of fluorescent proteins expressed in HEK cells completing one iteration directed evolution in a course of ~ 8 days. We employed this approach to develop a set of green and near-infrared fluorescent proteins with enhanced intracellular brightness. The developed near-infrared fluorescent proteins demonstrated high performance for fluorescent labeling of neurons in culture and in vivo in model organisms such as C.elegans, Drosophila, zebrafish, and mice. Spectral properties of the optimized near-infrared fluorescent proteins enabled crosstalk-free multicolor imaging in combination with common green and red fluorescent proteins, as well as dual-color near-infrared fluorescence imaging. The described method has a great potential to be adopted by protein engineers due to its simplicity and practicality. We also believe that the new enhanced fluorescent proteins will find wide application for in vivo multi-color imaging of small model organisms.

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Faculty Opinions recommendation of Efficient biotinylation and single-step purification of tagged transcription factors in mammalian cells and transgenic mice.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1014120.192900 ◽

2003 ◽

Author(s):

Ross Weatherman

Keyword(s):

Transcription Factors ◽

Transgenic Mice ◽

Mammalian Cells ◽

Single Step ◽

Single Step Purification

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A protocol for rapid degradation of endogenous transcription factors in mammalian cells and identification of direct regulatory targets

STAR Protocols ◽

10.1016/j.xpro.2021.100530 ◽

2021 ◽

Vol 2 (2) ◽

pp. 100530

Author(s):

Hillary M. Layden ◽

Nicholas A. Eleuteri ◽

Scott W. Hiebert ◽

Kristy R. Stengel

Keyword(s):

Transcription Factors ◽

Mammalian Cells ◽

Rapid Degradation

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DNA-binding specificity of GATA family transcription factors.

Molecular and Cellular Biology ◽

10.1128/mcb.13.7.3999 ◽

1993 ◽

Vol 13 (7) ◽

pp. 3999-4010 ◽

Cited By ~ 448

Author(s):

M Merika ◽

S H Orkin

Keyword(s):

Transcription Factors ◽

Dna Binding ◽

Mammalian Cells ◽

Chimeric Protein ◽

Binding Specificity ◽

Mobility Shift ◽

Binding Domains ◽

Dna Binding Specificity ◽

Mobility Shift Assay ◽

Gata Family

GATA-binding proteins constitute a family of transcription factors that recognize a target site conforming to the consensus WGATAR (W = A or T and R = A or G). Here we have used the method of polymerase chain reaction-mediated random site selection to assess in an unbiased manner the DNA-binding specificity of GATA proteins. Contrary to our expectations, we show that GATA proteins bind a variety of motifs that deviate from the previously assigned consensus. Many of the nonconsensus sequences bind protein with high affinity, equivalent to that of conventional GATA motifs. By using the selected sequences as probes in the electrophoretic mobility shift assay, we demonstrate overlapping, but distinct, sequence preferences for GATA family members, specified by their respective DNA-binding domains. Furthermore, we provide additional evidence for interaction of amino and carboxy fingers of GATA-1 in defining its binding site. By performing cotransfection experiments, we also show that transactivation parallels DNA binding. A chimeric protein containing the finger domain of areA and the activation domains of GATA-1 is capable of activating transcription in mammalian cells through GATA motifs. Our findings suggest a mechanism by which GATA proteins might selectively regulate gene expression in cells in which they are coexpressed.

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Maternal endotoxin exposure attenuates allergic airway disease in infant rats

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00399.2009 ◽

2010 ◽

Vol 298 (5) ◽

pp. L670-L677 ◽

Cited By ~ 18

Author(s):

Lei Cao ◽

Jinxia Wang ◽

Yingchun Zhu ◽

Irene Tseu ◽

Martin Post

Keyword(s):

Transcription Factors ◽

Cell Differentiation ◽

T Cell ◽

Airway Disease ◽

Positive Control ◽

Allergic Airway Disease ◽

T Helper ◽

T Helper 1 ◽

Pregnant Rats ◽

Neonatal Immune System

Prenatal exposures to immunogenic stimuli, such as bacterial LPS, have shown to influence the neonatal immune system and lung function. However, no detailed analysis of the immunomodulatory effects of LPS on postnatal T helper cell differentiation has been performed. Using a rat model, we investigated the effect of prenatal LPS exposure on postnatal T cell differentiation and experimental allergic airway disease. Pregnant rats were injected with LPS on day 20 and 21 (term = 22 days). Some of the offspring were sensitized and challenged with ovalbumin. Positive control animals were placebo exposed to saline instead of LPS, whereas negative controls were sensitized with saline. Expression of T cell-related transcription factors and cytokines was quantified in the lung, and airway hyperresponsiveness was measured. Prenatal LPS exposure induced a T helper 1 (TH1) immune milieu in the offspring of rats [i.e., increased T-bet and TH1 cytokine expression while expression of TH2-associated transcription factors (GATA3 and STAT6) and cytokines was decreased]. Prenatal LPS exposure did not trigger TH17 cell differentiation in the offspring. Furthermore, prenatal LPS exposure reduced ovalbumin-induced (TH2-mediated) airway inflammation, eosinophilia, and airway responsiveness. Thus, in utero exposure to endotoxin promotes a TH1 immune environment, which suppresses the development of allergic airway disease later in life.

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