Need a primary neuron transfection protocol

3 posts / 0 new
Last post
cdk5
cdk5's picture
Need a primary neuron transfection protocol

I would like know the best way to transfect primary neurons later than DIV10. I will appreciate if someone can help me out.

Fraser Moss
Fraser Moss's picture
This paper has a protocol for

This paper has a protocol for transfection of primary neurons at DIV 10 by calcium phosphate

Calcium influx via the NMDA receptor induces immediate early gene transcription by a MAP kinase/ERK-dependent mechanism.

Xia Z, Dudek H, Miranti CK, Greenberg ME.

Department of Neurology, Children's Hospital, Boston, Massachusetts 02115, USA.

The regulation of gene expression by neurotransmitters is likely to play a key role in neuroplasticity both during development and in the adult animal. Therefore, it is important to determine the mechanisms of neuronal gene regulation to understand fully the mechanisms of learning, memory, and other long-term adaptive changes in neurons. The neurotransmitter glutamate stimulates rapid and transient induction of many genes, including the c-fos proto-oncogene. The c-fos promoter contains several critical regulatory elements, including the serum response element (SRE), that mediate glutamate-induced transcription in neurons; however, the mechanism by which the SRE functions in neurons has not been defined. In this study, we sought to identify transcription factors that mediate glutamate induction of transcription through the SRE in cortical neurons and to elucidate the mechanism(s) of transcriptional activation by these factors. To facilitate this analysis, we developed an improved calcium phosphate coprecipitation procedure to transiently introduce DNA into primary neurons, both efficiently and consistently. Using this protocol, we demonstrate that the transcription factors serum response factor (SRF) and Elk-1 can mediate glutamate induction of transcription through the SRE in cortical neurons. There are at least two distinct pathways by which glutamate signals through the SRE: an SRF-dependent pathway that can operate in the absence of Elk and an Elk-dependent pathway. Activation of the Elk-dependent pathway of transcription seems to require phosphorylation of Elk-1 by extracellular signal-regulated kinases (ERKs), providing evidence for a physiological function of ERKs in glutamate signaling in neurons. Taken together, these findings suggest that SRF, Elk, and ERKs may have important roles in neuroplasticity.

PMID: 8757255

Some Other suggestions

This protocol is described for 8 DIV but you may be able to adapt it. https://www.roche-applied-science.com/PROD_INF/BIOCHEMI/no3_99/PG20.PDF

Also look in the following book

large version

Transfection Methods for Neurons in Primary Culture
Book Cellular and Molecular Methods in Neuroscience Research
Publisher Springer New York
DOI 10.1007/b98863
Copyright 2002
ISBN 978-0-387-95386-1 (Print) 978-0-387-22460-2 (Online)
DOI 10.1007/978-0-387-22460-2_3
Pages 29-36
Subject Collection Biomedical and Life Sciences
SpringerLink Date Friday, December 14, 2007

scisource
scisource's picture
Check out Cellectricon's

Check out Cellectricon's Systems at www.cellectricon.se

I had done BD for this company some time back and they were highly successful both with low throughput, high efficiency in adherent neurons and tissue sections as well as HT transfections in primary neurons for screening (also adherent). No trypsinizing your cells, no toxic chemical addition. It's a win-win. See attached.

Their systems are available now in the U.S. through SCiSOURCE.com