Never try to reach Eden

Without the guise of gifted intelligence, I'm going to portray a correct view of my world. 

I can feel the pressure, more then just the airspace above me, pressing down to crush my determined stance. Each day ticks away the moments that I share with sanity. I can see the stream of light from the other side widening with the waning weeks. It brings me closer, closer to a personal academic reform, comparative to Mao's Great Leap Forward. A movement of crushing proportions, contrived with the intention of breaking backs and depressing will until something of worth can be achieved. Tis the cycle, of life, death, and a yearning for knowledge. A little suffering must be in store, for the greater good. 

What is this greater good that calls for superficial sacrifice? It is a touch of my ambition, a taste of my determination to make an indentation, however little. This is what I have compiled for today. The fruits of my labour, I am to record before my silly little forgetful mind lingers in more trivial matters. 

Introduction:

 Using Light to facilitate Walking Movement by incorporating channel Rhodopsin in the neurons of the Spinal central Pattern Generator

Spinal cord transection, both complete and incomplete have been used to model the injuries sustained by patients who have lost locomotion (paraplegics). The specific location of the lower lumbar segments are of special interest to us, which points to the true location of the Central Pattern Generator (CPG). The CPG is a debated region of the lower Thoracic and upper Lumbar segments that acts as a neuro-network responsible for pattern and rhythm generation of the ANS, specifically in regards to walking movements. With the knowledge that the CPG can be stimulated by 5-HT, we can trace the location of the CPG by using immunihistochemistry to label areas of the spinal cord with 5-HT input. 

A minimally invasive technique that could restore nerve function is seen in the delivery of a light-gated protein to the injury site. We will explore the plausibility of inserting the gene for Channel Rhodopsin-2 via a viral vector into the spinal cord at the level of the CPG.In addition, Green fluorescence protein will be paired with ChR2 to visualize location. 

As of currently, our collaboration with Dr. Pan will reveal which two promoters will best fit our interest. Our optimal promoter should be specific for cells of the CPG. There has been approximately 22 different strains which have been investigated uses Cre/loxP recombination; a specific promoter attached to the Cre protein is crossed with the GFP protein with it's ubiquitous promoter. The Cre protein binds to the loxP sites. loxP is a 13 base pair inverted repeats with an 8 bp aysmmetric spacer. Cre excises at the location of the 8bp spacer to either eliminate or activate a gene. In our case, we seek to activate the expression of GFP by somehow disturbing the stop codon. 

That's all I've got. There are still some loose ends I need to tie up. For example: 

1. I need Dr. Pan's lab to explain to me how these two promoters work with one other, and does in fact Cre activate or eliminate? Or both in one sweeping blow?
   
2. Once we determine the promoters, how do they incorporate in the viral vector? 
3. Ultimately, what is the mechanism that is used to incorporate our target gene with GFP into the CPG? 

Other things to consider:

1. How do I use to fluorescent scope?
2. I need to research more into ChR2 and the adeno-associated viral vector. 
3. I need to understand the CPG's details like the back of my hand.

No time for expression of feelings today. I feel that if I start on the train of self expression, I may have no time for indifferent research. As much as I would like, this is not the location of my individual but one of professional strife. One other note, pod-stream is playing a playlist that mirrors my own collection. I must have good taste.