Calcium phosphate-mediated transfection of primary cultured brain neurons using GFP expression as a marker: application for single neuron electrophysiology

Watanabe, Shinju Y.; Albsoul-Younes, Abla; Kawano, Takeharu; Itoh, Hiroshi; Kaziro, Yoshito; Nakajima, Shigehiro; Nakajima, Yoshiaki

doi:10.1016/s0168-0102(98)00113-8

Cited by 28 publications

(18 citation statements)

References 23 publications

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“…The role of RhoA was analyzed with a dominant positive RhoA QL (33), dominant negative RhoA N19 (34), or the botulinum C3 exoenzyme, TC3 (35). In all cases cells were cotransfected with 1 g of expression vector encoding green fluorescent protein (GFP) (36). A group of cells was transfected with the corresponding empty vector as control.…”

Section: Methodsmentioning

confidence: 99%

Somatostatin Interferes with Thyrotropin-induced G1-S Transition Mediated by cAMP-dependent Protein Kinase and Phosphatidylinositol 3-Kinase

Medina

Toro

Santisteban

2000

Journal of Biological Chemistry

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kip1 . These correlated events are necessary for FRTL-5 cell proliferation after TSH stimulation. Moreover, TSH through PKA pathway increases cyclin-dependent kinase 2 levels, whereas PI 3-kinase signaling increases cyclin E levels. Together, both pathways finally converge, increasing the formation and activation of cyclin E⅐cyclin-dependent kinase 2 complexes and the phosphorylation of the retinoblastoma protein, two important steps in the transition from G 1 to S phase in growth-stimulated cells. Somatostatin exerts its antiproliferative effect inhibiting more upstream the TSH stimulation of PKA and PI 3-kinase, interfering with the TSH-mediated increases of intracellular cAMP levels by inactivation of adenylyl cyclase activity. Together, these results suggest the existence of a PKA-dependent pathway and a new PKA-independent PI 3-kinase pathway in the TSH/cAMP-mediated proliferation of FRTL-5 thyroid cells.

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Section: Methodsmentioning

confidence: 99%

Somatostatin Interferes with Thyrotropin-induced G1-S Transition Mediated by cAMP-dependent Protein Kinase and Phosphatidylinositol 3-Kinase

Medina

Toro

Santisteban

2000

Journal of Biological Chemistry

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“…The transfection rate was 60.2% for the entire coverslip, far more than the usual 1-5% transfection efficiency reported in the literature. 3,8,9 Our routine transfection efficiency usually falls in the range of 25-40% (n410). Typically, the earliest GFP fluorescence signal was observed in cell bodies within 4 h after transfection (dissolving precipitate).…”

Section: High Transfection Efficiency In Hippocampal Microisland Cultmentioning

confidence: 99%

“…Interestingly, very few glial cells were transfected comparing to the number of neurons transfected in our cultures, consistent with previous findings. 9 This is possibly because the monolayer of astrocytes were treated with Ara-c (cytosine-barabinofuranoside, 4 mM) to stop their overproliferation before plating neurons on top of them. It is interesting to note that much more glial cells will be transfected before Ara-C treatment.…”

Section: High Transfection Efficiency In Hippocampal Microisland Cultmentioning

confidence: 99%

“…[1][2][3] There have been many attempts in various labs to improve the Ca 2+ -phosphate transfection efficiency in neurons. 8,9 However, the highest efficiency reported so far is 13.5%, 8 far less than other methods such as viral infection, which often reaches more than 40% of transduction efficiency. 10,11 To increase the transfection rate while maintaining low toxicity is the ultimate goal for every gene transfer method.…”

Section: Introductionmentioning

confidence: 99%

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High Ca2+-phosphate transfection efficiency enables single neuron gene analysis

2004

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“…These goals are especially difficult to achieve for postmitotic primary neurons due to their high sensitivity to any microenvironmental change [1][2][3][4][5] . Many currently available transfection methods do not yield sufficiently good gene expression results to allow functional gene analysis in differentiated adherent neurons in vitro.…”

Section: Introductionmentioning

confidence: 99%

Efficient transfection of DNA or shRNA vectors into neurons using magnetofection

et al. 2007

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Efficient and long-lasting transfection of primary neurons is an essential tool for addressing many questions in current neuroscience using functional gene analysis. Neurons are sensitive to cytotoxicity and difficult to transfect with most methods. We provide a protocol for transfection of cDNA and RNA interference (short hairpin RNA (shRNA)) vectors, using magnetofection, into rat hippocampal neurons (embryonic day 18/19) cultured for several hours to 21 d in vitro. This protocol even allows double-transfection of DNA into a small subpopulation of hippocampal neurons (GABAergic interneurons), as well as achieving long-lasting expression of DNA and shRNA constructs without interfering with neuronal differentiation. This protocol, which uses inexpensive equipment and reagents, takes 1 h; utilizes mixed hippocampal cultures, a transfection reagent, CombiMag, and a magnetic plate; shows low toxicity and is suited for single-cell analysis. Modifications done by our three laboratories are detailed. INTRODUCTIONEvery gene transfer method aims to achieve high transfection efficiency, low toxicity and long-lasting expression. These goals are especially difficult to achieve for postmitotic primary neurons due to their high sensitivity to any microenvironmental change 1-5 . Many currently available transfection methods do not yield sufficiently good gene expression results to allow functional gene analysis in differentiated adherent neurons in vitro. A recent report provides a useful overview of seven different gene delivery methods, including two often-used transfection techniques (calcium-phosphate and lipofection), but excluding magnetofection 6 . Magnetofection is a technique that can be used to reliably and efficiently introduce DNA into a variety of cell types 7 . Therefore, we have invested in designing a reliable, magnetofection-based protocol to express DNA or shRNA constructs in rat hippocampal neurons (embryonic day (E)18/19) cultured for several hours to 21 d in vitro (DIV). After transfection, neurons expressing exogenous proteins can develop normally in culture for 5-10 d. We have determined the optimum parameters for transfection efficiency such as age of cells at transfection time point, expression levels during the time after transfection and parameters for doubletransfection. This protocol shows low toxicity, uses a combination of a transfection reagent, CombiMag, and magnetic force and shows a sufficiently high efficiency in neurons (ca. 5%) to allow single-cell analysis.We present a core protocol and illustrate the critical steps, modifications and applications carried out by our three laboratories (Medina, Fuhrer and Fritschy) [8][9][10] . This protocol allows single and double transfections of DNA vectors at different time points after plating. First, we present a detailed characterization of magnetofection parameters such as age of neurons, time course of protein expression after magnetofection, and transfection efficiency in pyramidal neurons and GABAergic interneurons. Three specific

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Calcium phosphate-mediated transfection of primary cultured brain neurons using GFP expression as a marker: application for single neuron electrophysiology

Cited by 28 publications

References 23 publications

Somatostatin Interferes with Thyrotropin-induced G1-S Transition Mediated by cAMP-dependent Protein Kinase and Phosphatidylinositol 3-Kinase

Somatostatin Interferes with Thyrotropin-induced G1-S Transition Mediated by cAMP-dependent Protein Kinase and Phosphatidylinositol 3-Kinase

High Ca2+-phosphate transfection efficiency enables single neuron gene analysis

Efficient transfection of DNA or shRNA vectors into neurons using magnetofection

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