DNA Transfection: Calcium Phosphate Method

Kwon, Munjin; Firestein, Bonnie L.

doi:10.1007/978-1-62703-444-9_10

Cited by 69 publications

(44 citation statements)

References 8 publications

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“…The Pcdh19ECFc fusion constructs were transfected alone or with NcadEC W2A/R14E His into HEK293 cells using calcium-phosphate transfection (Kwon and Firestein, 2013; Barry et al, 2014; Jiang and Chen, 2006). Briefly, solution A (10 µg of plasmid DNA and 250 mM CaCl 2 ) was added drop-wise to solution B (2x HBS) while mildly vortexing, and the final transfection solution was added drop-wise to two 100 mm dishes of cultured HEK293 cells.…”

Section: Materials and Methodsmentioning

confidence: 99%

Structural determinants of adhesion by Protocadherin-19 and implications for its role in epilepsy

Cooper

Jontes

Sotomayor

2016

eLife

116

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Non-clustered δ-protocadherins are homophilic cell adhesion molecules essential for the development of the vertebrate nervous system, as several are closely linked to neurodevelopmental disorders. Mutations in protocadherin-19 (PCDH19) result in a female-limited, infant-onset form of epilepsy (PCDH19-FE). Over 100 mutations in PCDH19 have been identified in patients with PCDH19-FE, about half of which are missense mutations in the adhesive extracellular domain. Neither the mechanism of homophilic adhesion by PCDH19, nor the biochemical effects of missense mutations are understood. Here we present a crystallographic structure of the minimal adhesive fragment of the zebrafish Pcdh19 extracellular domain. This structure reveals the adhesive interface for Pcdh19, which is broadly relevant to both non-clustered δ and clustered protocadherin subfamilies. In addition, we show that several PCDH19-FE missense mutations localize to the adhesive interface and abolish Pcdh19 adhesion in in vitro assays, thus revealing the biochemical basis of their pathogenic effects during brain development.DOI: http://dx.doi.org/10.7554/eLife.18529.001

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Section: Materials and Methodsmentioning

confidence: 99%

Structural determinants of adhesion by Protocadherin-19 and implications for its role in epilepsy

Cooper

Jontes

Sotomayor

2016

eLife

116

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“…Briefly, neurons were plated onto 300, 600, and 3,000 Pa gels. On day in vitro (DIV) 7 or 8, neurons were transfected with pEGFP for dendrite visualization using the calcium phosphate method . On DIV10, cultures were treated with vehicle control or with 10 µM L‐glutamate.…”

Section: Methodsmentioning

confidence: 99%

“…On day in vitro (DIV) 7 or 8, neurons were transfected with pEGFP for dendrite visualization using the calcium phosphate method. 19 On DIV10, cultures were treated with vehicle control or with 10 mM L-glutamate. Neurons were fixed on DIV 12, imaged at 2003 using an Olympus Optical IX50 microscope (Tokyo, Japan) with a Cooke Sensicam CCD cooled camera, fluorescence.…”

Section: Hippocampal Neuron Culturementioning

confidence: 99%

Glutamate affects dendritic morphology of neurons grown on compliant substrates

Previtera

Firestein

2015

Biotechnology Progress

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Brain stiffness changes in response to injury or disease. Similarly, as a secondary consequence, glutamate is released from neurons and astroglia. Two types of glutamate receptors, N-methyl-D-aspartate (NMDA) and α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, sense mechanotransduction, leading to downstream signaling in neurons. Recently, our group reported that these two receptors affect dendrite morphology in hippocampal neurons grown on compliant substrates. Blocking receptor activity has distinct effects on dendrites, depending on whether neurons are grown on soft or stiff gels. In the current study, we examine whether exposure to glutamate itself alters stiffness-mediated changes to dendrites in hippocampal neurons. We find that glutamate augments changes seen when neurons are grown on soft gels of 300 or 600 Pa, but in contrast, glutamate attenuates changes seen when neurons are grown on stiff gels of 3000 Pa. These results suggest that there is interplay between mechanosensing and glutamate receptor activation in determining dendrite morphology in neurons.

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“…siRNAs were transfected using calcium phosphate transfection method [85]. siRNAs were transfected using calcium phosphate transfection method [85].…”

Section: Sirna Transfectionsmentioning

confidence: 99%

“…Small interfering RNA oligonucleotides were purchased from MWG/ Eurofins and used in a stock concentration of 20 lM. siRNAs were transfected using calcium phosphate transfection method [85]. siRNA oligonucleotides were diluted in a solution containing distilled sterile H 2 O and 2M CaCl 2 .…”

Section: Sirna Transfectionsmentioning

confidence: 99%

CDK 1 and CDK 2 regulate NICD 1 turnover and the periodicity of the segmentation clock

et al. 2019

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All vertebrates share a segmented body axis. Segments form from the rostral end of the presomitic mesoderm (PSM) with a periodicity that is regulated by the segmentation clock. The segmentation clock is a molecular oscillator that exhibits dynamic clock gene expression across the PSM with a periodicity that matches somite formation. Notch signalling is crucial to this process. Altering Notch intracellular domain (NICD) stability affects both the clock period and somite size. However, the mechanism by which NICD stability is regulated in this context is unclear. We identified a highly conserved site crucial for NICD recognition by the SCF E3 ligase, which targets NICD for degradation. We demonstrate both CDK1 and CDK2 can phosphorylate NICD in the domain where this crucial residue lies and that NICD levels vary in a cell cycle‐dependent manner. Inhibiting CDK1 or CDK2 activity increases NICD levels both in vitro and in vivo, leading to a delay of clock gene oscillations and an increase in somite size.

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DNA Transfection: Calcium Phosphate Method

Cited by 69 publications

References 8 publications

Structural determinants of adhesion by Protocadherin-19 and implications for its role in epilepsy

Structural determinants of adhesion by Protocadherin-19 and implications for its role in epilepsy

Glutamate affects dendritic morphology of neurons grown on compliant substrates

CDK 1 and CDK 2 regulate NICD 1 turnover and the periodicity of the segmentation clock

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