Dp260 Creating a subject to study: Viral vector is okay, just inject a lot of it and see what happens.

. This will bind to every type of muscle (cardiac, skeletal, etc.) and other tissues such as the liver (which is a problem) Use a specific heart promoter (creatin/kinase?) for expression in heart Dystrophin is too large to be carried by a viral vector, so you would need to insert two pieces in a different viral vector and have them homologously recombine due to overhanging ends. Better idea: Create a transgenic mouse because dystrophin is so large. Use alpha myosin heavy chain promoter Problem: Transgene construct will insert randomly into genome Solution: Make multiple different zygotes that will insert in different locations to find which one is successful Things to watch for: 1. How many copies of the transgene get into each genome will impact the overall expression level of Dp260. 2. If it inserts in a bad spot (either a spot that disrupts another gene or a spot that is only active in reproduction, etc.), then the gene will not be expressed. Transgenic mouse WILL express Dp260 only in heart cells if heart-only promoter is used, so we will be able to tell whether the insertion of Dp260 in mdx mice restores the function or not. Potential extension: See what expression in skeletal AND cardiac muscle does. How will we measure heart functioning? And Why? The lifespan of mdx and WT mice is similar, so seeing whether they die or not NATURALLY will not work very well. To fight this, you have to stress the mice and make them work harder. Two ways: Adenergic stimulation Pump adrenaline into them in one of two ways to stress out and wear out heart earlier. Shows how effectively heart manages stress, a key to heart functioning. Can either pump in one big dose of a drug (such as beta-1 adenergic receptor) that causes heart to contract more vigourously, OR insert small dose that damages heart but allows it to survive allows for longer observation Can use sponge semipermeable membrane contraption (see Alzet website) that is

Ways to test heart for effectiveness: Echocardiogram - basically a ultrasound for the heart PV loops Measures systolic/diastolic heart beats, and will determine how successful the heart is at beating by measuring the volume and pressure of the heart beating Can be graphed together, creating a loop Then, you either load or unload the heart to see what changes (while still graphing) Load - pump more blood into heart manually (through aorta), which causes a strong contraction Heart has mechanism that detects a greater volume of blood and increases the size of the contraction to match it. Use this naturally occurring mechanism to manipulate pressure and volume graph. Unload- block vena cava by clamping it off (like a garden hose that you step on) Similarly, heart weakens its contraction when less volume is inside, so use this to graph another circle As you do this, use the different graphs to generate a line connecting the different points. This line is used to determine how effective the heart is functioning. Can also measure heart wall thickness from this analysis Also, can compare different graphs to determine which hearts are functioning better Another way to test is by testing leakage in heart cells Sarcomeres (muscle fiber bundles that contract) are attached to dystrophin. dystrophin then coils and attaches to the plasma membrane When the sarcomere contracts, the dystrophin expands, absorbing the force, rather than the cell wall In dystrophy, sarcomeres attach to plasma membranes, causing holes when contracting. This can be tested by staining heart cells for substances that shouldnt normally be in heart cells (IgG or Albumin/Evans Blue work) So What exactly are we testing? We are testing whether Dp260 recovers any of the function of dystrophin in mdx (dystophin knockout mice) by creating a transgenic mouse that contains a functional Dp260 gene. The results will be obtained by analyzing heart cells for

soaked in dobutamine or isoproterol (both andrenic stimulantors) Uses osmosis to release hormone

leakage, echocardiograms, and PV loops.

Dp40 The shortest forms of dystrophin (Dp 71/58/40) all are not structural proteins, unlike the other longer forms. This means that it plays a different role than other forms (likely.. cant be certain but they do know that it is not a structural protein) Potentially a signalling protein or some other function The two myosin binding sites found in dystrophin are BEFORE the Dp71 promoter, suggesting it is not a protein that binds to muscles Create knockout of short isoforms of dystrophin (Dp71, 58, 40) If we are going to knockout all 3, we can just mess with the promoter of these These have their own promoter within dystrophin Dystrophin has a few embedded promoters Potentially could knock down (knock down is correct terminology when not knocking out entire gene) these 3 by removing the ATG start codon. Shouldnt mess with dystrophin protein as a whole If we dont want to knockout all three, we would have to make a special knockout that takes a little more work Either use siRNA or Antisense bonding to destroy mRNA Works by binding to the 3UTR of Dp40 (which isnt very large so I am not exactly sure what sequence would work) When it binds, your cell will have a double stranded DNA segment, which then puts up a red flag that it is a virus. So, the cell destroys the mRNA (thus destroying Dp40) siRNA works slightly differently, causing stuff to bind to the siRNA (which is like 24bp) and creates a glob that is destroyed by the cell Ways to measure Dp40/58/71 Use electrochemistry to see how much neurotransmitter (5-HT (serotonin), norepinephrine, histamine) is released in synapse Literally put probe in synapse that measures electrical current Can tell difference between neurotransmitters and can tell how much is released Also, fluorescent tagging works Put a bunch of FM1-43 fluorescent tag on the cells, which will coat the plasma membrane Wash away excess (but the coating on the membrane will remain) When vesicles bind to the membrane, they fuse to it. Since membranes are not stained, a gap will show up where vesicle fused As time passes, you can monitor the amount the fluorescence gets spread out (both quantitatively and qualitatively (fluorescence values vs. what each membrane looks like)

Should be able to graph it Another thing that works is the patching a neuron Put a patch on the neuron that can stimulate a neuron at will in vitro typically Can stimulate it to measure electrochemically how the neuron functions What are we testing and measuring? The goal of this is to study the role of Dp40 (or the short isoforms of dystrophin) by creating a knockdown mouse. Results will be gathered by electrochemical analysis of the neuron and synapse, cell membrane fluorescence, and neuron patching.

Outline of Introduction: (Must be around 3 pages) What is known about the structure of your protein? Where in the cell does your protein function, and what is known about its function? What species or groups of species have genes that encode your protein? Is your protein part of a large family of evolutionarily related proteins? What is still not understood about your protein and its function? If your proposed experiments are based on previous work by other researchers, this earlier work should be described.

Outline of Impact Statement:: (1 page double spaced) Why is further research on your protein important? A better understanding of your protein may contribute to our basic knowledge of cell biology. Mutant versions of your protein may cause disease. Your protein may the target of drugs that are used to treat disease. Your protein may have biotechnological applications.