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.

Sunday, August 24, 2008

Down Regulating the Smad and Neuro-regeneration

The inventors down regulated Smad 2/3 (an inhibitor of neuro-regeneration) in vivo via delivery of siRNA to the spinal cord using catheters.

Inhibiting smad signaling promotes neuron regeneration.
Inventors: Fan Wang, Zhigang He
USPTO Application #: 20080031911
Inhibition of Smad2/3 Signaling Promotes Axonal Regeneration after Spinal Injury in Rats-Gene Expression Knockdown in vivo!
One hour after the spinal cord is lesioned, the rats in the SB-505124 group receive a bolus injection of SB-505124 (30 mg/kg) in 0.9% saline administered via a tail vein. The treatment is repeated every 24 hours on days 1 through 7 post-lesion. Vehicle only control rats undergo the same treatment but are injected with an equal volume of 0.09% saline in a tail vein. At the same treatment time points as the SB-505124 group, the Smad2/3 siRNA group and corresponding controls receive 10 .mu.l rat Smad2/3 siRNA (Dharmacon, Lafayette, Colo.), mismatch siRNA, or transfection reagent only delivered to the spinal cord via the catheters. The siRNA (or mismatch siRNA control) complexes are prepared immediately prior to administration by mixing the RNA solution (200 .mu.M in annealing buffer) with a transfection reagent, i-Fect ™ . (Neuromics, Edina, Minn.), in a ratio of 1:4 (w:v). At this ratio, the final concentration of RNA as an RNA/lipid complex is 2 .mu.g in 10 .mu.l.