Thursday, August 28, 2008

Transfecting Sympathetic Neurons

GDNF and Ret are important to the growth, maintenance and survival of Neurons. The GDNF ligands act via activation of Ret. Every step towards understanding the intricacies of this pathway, brings researchers closer towards unlocking the code for Neurodegenerative Disease therapies.

siRNA is an important tool for studying the Neurotrophic pathways as researchers can use it to modulate the expression of related receptors. The tricky part is getting sufficient siRNA into neurons to do the appropriate studies of how modulating targeted genes results in changes in protein expression.

Here Drs. Cynthia Tsui and Brian Pierchala have published results from there studies of C2AP and Cbl-3/Cbl-c and Ret Transduction. One of the keys to this study was using siRNA to silence CD2AP and Cbl-3 expression. By turning these off they were able to identify a critical checkpoint in the Ret pathway.

Cynthia C. Tsui and Brian A. Pierchala CD2AP and Cbl-3/Cbl-c Constitute a Critical Checkpoint in the Regulation of Ret Signal TransductionJ. Neurosci., Aug 2008; 28: 8789 - 8800 ; doi:10.1523/JNEUROSCI.2738-08.2008.

...Control, CD2AP, and Cbl-3 siRNAs (Applied Biosystems/Ambion) were transfected into 4 DIV sympathetic neurons using the i-Fect ™ reagent according to the manufacturer’s instructions (Neuromics). Transfection efficiency was determined by the cotransfection of a fluorescently labeled nontargeting, control siRNA (siGLO RISC-free siRNA; Dharmacon RNA Technologies). Immunoblotting of the targeted proteins determined that the maximal knockdown of protein expression was observed 72 h after siRNA transfection. Greater than 90% of SCG neurons were transfected, as ascertained by the level of intracellular fluorescence of the siGLO siRNA.

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