Optimized PEI‐based Transfection Method for Transient Transfection and Lentiviral Production

Yang, Shaozhe; Zhou, Xiaoling; Li, Rongxiang; Fu, Xiuhong; Sun, Pingnan

doi:10.1002/cpch.25

Cited by 33 publications

(26 citation statements)

References 28 publications

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“…MV D7 cells were maintained in a 32 °C humidified incubator under 5% CO 2 . For lentiviral production, second generation lentiviral particles were generated by PEI transfection of 293T cells as previously described (63) with transfer plasmid, pMD2.G, and psPAX2 (Addgene #12259, #12260, gifts from Didier Trono). HEK293T media containing lentiviral particles was collected, filtered, and added directly to cultures with polybrene (Gibco).…”

Section: Methodsmentioning

confidence: 99%

Native proline-rich motifs exploit sequence context to target actin-remodeling Ena/VASP proteins

Hwang

Grant

Ilunga

et al. 2021

Preprint

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Protein interactions between intrinsically disordered, short linear motifs (SLiMs) and modular recognition domains are critical elements in many signal transduction pathways. Yet for most interactions, it is still unclear how low-complexity SLiMs discriminate between highly conserved but biologically distinct SLiM-binding proteins. The Ena/VASP family of proteins pose one such specificity problem. Paralogs ENAH, VASP, and EVL bind to a 5-residue SLiM prevalent in the proteome and perform distinct cellular functions despite sharing high sequence and structural similarity. To interrogate how the sequence context of SLiMs impacts Ena/VASP interactions, we performed an unbiased proteomic screen against the ENAH EVH1 domain. We discovered unexpected ways in which local and distal sequence elements flanking native SLiMs modulate binding. Particularly notable is a peptide from PCARE that achieves paralog specificity by stabilizing a unique conformation adopted only by ENAH. A PCARE-derived peptide can selectively recruit ENAH and block its activity in cells, establishing it as a valuable reagent to disentangle the roles of Ena/VASP paralogs and a potential agent for modulating ENAH function in breast cancer. Guided by our analyses of native interactions, we designed the tightest known ENAH EVH1 binder with a dissociation constant of 50 nM and 400-600-fold selectivity over EVL and VASP. Our work demonstrates that sequence context plays a prominent role in dictating SLiM interactions and can inform the design of custom molecules that target SLiM-based interactions.

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Section: Methodsmentioning

confidence: 99%

Native proline-rich motifs exploit sequence context to target actin-remodeling Ena/VASP proteins

Hwang

Grant

Ilunga

et al. 2021

Preprint

View full text Add to dashboard Cite

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“…For exogenous protein expression in cultured neurons, second generation lentiviral particles were generated by PEI transfection of 293T cells as previously described ( Yang et al, 2017 ) with transfer plasmid, pMD2.G, and psPAX2 (Addgene #12259, #12260, gifts from Didier Trono). At 24 and 48 h post-transfection, 293T media containing lentiviral particles was collected, combined, filtered, and added directly to neuron cultures without polybrene at the time of plating.…”

Section: Methodsmentioning

confidence: 99%

High Fidelity Cryopreservation and Recovery of Primary Rodent Cortical Neurons

Parker

Moutal

Cai

et al. 2018

eNeuro

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Cell cryopreservation improves reproducibility and enables flexibility in experimental design. Although conventional freezing methodologies have been used to preserve primary neurons, poor cell viability and reduced survival severely limited their utility. We screened several high-performance freezing media and found that CryoStor10 (CS10) provided superior cryoprotection to primary mouse embryonic cortical neurons compared to other commercially-available or traditional reagents, permitting the recovery of 68.8% of cells relative to a fresh dissection. We characterized developmental, morphometric, and functional indicators of neuron maturation and found that, without exception, neurons recovered from cryostorage in CS10 media faithfully recapitulate in vitro neurodevelopment in-step with neurons obtained by fresh dissection. Our method establishes cryopreserved neurons as a reliable, efficient, and equivalent model to fresh neuron cultures.

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“…Cells are maintained at 37°C and 5% CO2. 60% confluent HEK293T cells were transfected using PEI (Polysciences, 23966) in OptiMEM (Thermo, 31985070) as previously described (Yang et al, 2017) with transfer plasmid, pMD2.G, and psPAX2 (gifts from Didier Trono, Addgene #12259 and #12260; RRID:Addgene_12259 and RRID:Addgene_12260) at a 1:0.25:0.75µg plasmid ratio and 3:1 PEI:DNA ratio. Viral supernatant was collected 48-72 hours post-transfection, filtered through 0.45µm filters, and added directly to primary neuron cultures.…”

Section: Cell Culturementioning

confidence: 99%

EVL and MIM/MTSS1 regulate actin cytoskeletal remodeling to promote dendritic filopodia in developing neurons

Grant

et al. 2021

Preprint

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Dendritic spines are the postsynaptic compartment of a functional neuronal synapse, and are critical for synaptic connectivity and plasticity. The developmental precursor to dendritic spines, dendritic filopodia, are highly motile protrusions that facilitate synapse formation by sampling the environment for suitable axon partners during development and learning. Despite the significance of the actin cytoskeleton in driving these protrusions, the actin remodeling factors involved in this process are not fully characterized. In this work, we identify a critical function for the Ena/VASP protein EVL in the regulation of dendritic filopodia. Amongst the Ena/VASP proteins, EVL is uniquely required for the characteristic morphology and dynamics of dendritic filopodia. Using a combination of genetic and optogenetic manipulations, we demonstrate that EVL promotes protrusive motility through membrane-direct actin polymerization at dendritic filopodia tips. EVL forms a complex at nascent protrusions and dendritic filopodia tips with MIM/MTSS1, an I-BAR protein recently discovered to be important for initiation of dendritic filopodia. We propose a model in which EVL cooperates with MIM to elongate and coalesce branched actin filaments, establishing the dynamic lamellipodia-like architecture of dendritic filopodia in developing neurons.

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Optimized PEI‐based Transfection Method for Transient Transfection and Lentiviral Production

Cited by 33 publications

References 28 publications

Native proline-rich motifs exploit sequence context to target actin-remodeling Ena/VASP proteins

Native proline-rich motifs exploit sequence context to target actin-remodeling Ena/VASP proteins

High Fidelity Cryopreservation and Recovery of Primary Rodent Cortical Neurons

EVL and MIM/MTSS1 regulate actin cytoskeletal remodeling to promote dendritic filopodia in developing neurons

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