Despite the imperfect animal model and lack of specific treatment for RP, there have been certain methods that have been tested for retinitis pigmentosa, including gene therapy, small molecules that stabilize the protein and microchips that take place of retinas. However, microchips that are currently in development would only be viable for those who have lost their sight completely. Those who still retain their eyesight does not have many treatment options. One treatment option for those people who still retain some eyesight is gene therapy. Because the eye is immunologically isolated from the rest of the body and easily accessible, some complications from gene therapy is not a factor in the eye. In the research paper, Cas9/sgRNA selective targeting of the P23H Rhodopsin mutant allele for treating retinitis pigmentosa by intravitreal AAV9.PHP.B-based delivery, the paper aimed at validating a CRISPR/Cas9 strategy to specifically inactivate the P23H RHO mutant while preserving the WT allele in vitro and translate the approach in vivo by delivering the CRISPR/Cas9 component in RHO mutant retina. In the first experiment that was done, the researchers created various Cas 9 containing vectors to test with one disrupted the P23H mutation most efficiently. The cleavage efficiency was measured using T7El assay, TIDE, and Sanger sequencing. The result indicated that sgRNA-mMUT discriminated with the highest specificity of the mutant RHO sequence, and no off-target indel was detected. The result indicates that sgRNA-mMUT is the best candidate and should be used in further studies regarding the gene therapy of the retina. In the second experiment, to test whether the treatment would work in vivo, the vectors were inserted into the retina of the mice by in vivo electroporation and the product of the genes was assessed for the behavior of the RHO protein and mutation rate in vivo. The final result of the experiment is that Cas9-VQR/sgRNA-mMUT system enabled an efficient and selective targeting of the P23H mutant capable of a high degree of inactivation in the RHO allele in vivo.
To measure the effect that thyroid hormone (TH) has on neural stem cells, thyroid hormones in zebrafish were manipulated, fixed, treated with EdU, and imaged. TH levels in zebrafish were altered by adding either, T4, a precursor to the T3 thyroid hormone that is biologically active, or PTU, which blocks the conversion of T4 to T3, therefore significantly decreasing the amount of thyroid hormones available to the body. In the first trial of the experiment we discovered that our drug dosage was too high and would kill the fish, so we lowered the doses in the second trial to 300nM of PTU and .5mM of T4. The 5dpf zebrafish were treated with respective drugs for two days. At 7dpf, the fish were fixed in 4% PFA and the brains were dissected. The dissected brains then went through an EdU Click-it copper-catalyzed cycloaddition reaction where a thiamine analog is added to dividing cells and labels those cells with fluorescent dye. EdU is incorporated into actively dividing cells and is often used to label stem cells in a proliferative state. The brains were then divided into well reacted and poorly reacted and imaged in both bright field and fluorescent field and merged. The representative images of each treatment in well reacted brain are shown in Figure 1. The poorly reacted brain are not shown because the poorly reacted brain could nor be imaged due to time constraints. Because of this, there are no poorly reacted brain EdU+ cell count data for this group.
The EdU positive cells in Lateral Recess and Posterior Recess were then counted and the number was recorded (table 1). The average and standard deviation was calculated for each condition. The result indicated that the average EdU positive cells in Lateral recess in the control is about 35( standard deviation:22), in PTU, 27, and in T4, 44(.7). Meanwhile the EdU positive cells in posterior recess is about 11(7.37) in control 15, in PTU and 13(1.17) in T4 (figure 2, A). The standard deviation was not included due to low sample size. While there are some differences between the different values, due to the low number of samples that are available, no significant conclusion can be reached with this result.
In the data which was gathered by the entire class, the well reacted brain data indicates that in the lateral recess, there is an average of 56(32) EdU+ cells in control, 62(37) in PTU treated cells, and 67(32) in T4 treated cells. This indicates that both PTU and T4 increases the amount of neural stem cell in the lateral recess. In the posterior recess., for controlling the average number of EdU+ cell is about 21(8), in PTU is about 21(5), and in T4 is 24(6) (figure 1, B). This indicates that while PTU does not have an effect on the number of neural stem cells, the addition of T4 increases the number of neural stem cells. However, the data may not be reliable since the standard deviation for the data is high. In the poorly reacted brains, the data indicates that there is an average of 38(21)for control, and 59 for PTU in the lateral region and 18 (7) for control and 23.5 for PTU in the posterior region (Figure 2, C). The standard deviation for PTU treated brains and the average and standard deviation of T4 treated cells were not included due to incomplete data. However, very few things are able to be concluded from the pooly reacted data because it is unknown whether the poor reaction is due to the cells having low neural stem cells or not reing reacted completely to the click it reaction, and there are very small sample size.
Alzheimer's disease is a neurodegenerative disease that typically affects adults over 65 years old. The disease is caused by abnormally high levels of beta amyloid protein and tau protein. The beta amyloid protein clusters in high numbers between neurons and eventually impede the signaling of the neurons, leading to death of the neurons. The tau proteins structure the microtubules in the axons. In alzheimer's the tau proteins deform and they impede the trasnport of molecules form the soma to the axon terminal. This also eventually impedes the signaling of the neurons and causes neuron death. Late onset alzheimer's disease has a genetic pre disposition involed in it, unlike early onset alzheimer's where there is a genetic mutation that can be passed from generation to generation. In late onset alzheimer's disease, the e4 allele of the APOE gene causes the pre disposition to the disease. The APOE gene is involved in trasnporting cholesterol and lipoproteins. It is believed that this allele in the APOE gene is a predisposition to cardiovascular disease as well as alzheimer's. cholesterol is mainly used by astrocytes which would ideally remove the amyloid plaques, but they don't in alzheimer's patients.
Prior to the methods project, I have never had an assignment where we were to write the way we did. It was tough at first to give direction in my methods project without telling the reader what I actually did to get the result I did. With practice in writing my drafts, I began to find the methods project running smoothly. Although there was a lot of out of class work to do, the project was very interesting to see how well I could follow someone’s methods to recreate a multi-panel scientific panel I have never seen before. Surprisingly, the end result for most of the classes replicate figure looked very close to the original. There were plenty of differences in each replicate, but for the most part, most replicates looked similar to the original. This meant that I was able to write in a way that others were able to understand and follow. The methods project allowed me to practice writing a methods section in the future for a scientific paper.
At first, I did not know what to write my perfect paragraphs about. I did not feel like I was improving my writing at first when I began to write these perfect paragraphs. I felt like I was just writing to get the assignment done. About a third into the semester, I changed my mindset about what I would write about and how well I wrote these paragraphs. I focused more on spelling, grammar and the actual construct of the paragraph to make it the best I could. This allowed my classmates to carefully read these paragraphs and offer constructive criticism for me to work on in my next perfect paragraph. I had never written like this before taking this class. This class was an eye opener in to how to properly write in science. The total effect this part of the class had on me was to make sure I write everything I write with no spelling and grammar errors.
Before beginning to write the daily drafts, I thought to myself that writing for 30 minutes a day is not long at all. However, soon after the semester began, I found myself slacking on writing my daily drafts. Soon after, I began to designate a time each day to spend 30 minutes writing. After writing a few of these drafts, I thought it was very interesting as it allowed us to write about almost anything I want. I found myself looking up articles about daily news in science, reading them, and writing a short summary about them. There were thousands of articles to choose from and I picked the ones that I thought were interesting. The good thing about this is that I enjoyed what I was reading so I can write about it a lot easier. Writing these drafts meant that I was improving the way I write and allowed me to understand scientific articles. Throughout the semester, these blog drafts had an effect on the way I was writing in science later on in the semester.
In genetics labs plasmids are a very valuble tool. Plasmids are circular strands of DNA that are found in bacteria. Plasmids must contain an origin of replication (where DNA polymerase binds), a multiple cloning site (where you can add any gene you want), and a selectable marker (usually an antibiotic resistant gene). These plasmids are used to inserted a selected gene into any cell. If you want a cell to contain a certain gene, you place that gene in the plasmid, then place the plasmid in the cell. The selectable marker is usually an antibiotic resistant gene because this allows you to select for cells that contain the plasmid. You place the cells you believe to have taken up the plasmid on a plate with an antibiotic of your choosing. Only the cells containing the plasmid, with antibiotic gene and the gene of your choosing, will be able to grow on this plate.
Embryosis is the formation of an embryo. There are two main steps to this process: blastulation, and gastrolation. The sperm and egg cell must fuse to form a zygote. The fusion of sperm and egg allow the genetic material to merge, all cells are pluripotent at this point. Clevage, compaction, and differentiation follow which form the blastocyte. The overall size of the blastocyte is not much bigger than the zygote, due to compaction. There is differentiation between cells in the blastocyte at this point. Gastrolation is the formation of three distinct layers in the blastocyte which will differentiate into different tissues in the bdoy. The top layer is the ectoderm, the middle layer is the mesoderm, and the bottom layer is the endoderm. The ectoderm differentiates into the nervous system and the skin. The mesoderm differentiates into the muscles; the endoderm differentiates into the internal organs. This process is virtually the same for all mamals. The outcome is very different because of the genetic information fused, resulting in a wide range of organisms.
The Methods project was a really interesting project. I was a bit confused in the beginning on how it would work and what I needed to do, but once I got the hang of the assignment I was really into it. This project helped me tremendously in my other classes, because I had the ability to practice writing a methods and collecting/presenting images. I also enjoyed the presentations; it made me work on my ability to present in front of a class. I really enjoyed this project and I think it aided in my research writing.
The Perfect Paragraphs left me with the same feeling as the drafts assignment. Initially, I thought it would not be a difficult assignment but I did struggle. I think that I started completing this assignment more thoroughly toward the middle of the semester. I would set up notifications and rewrite this assignment in my planner to help remind me. I found that this assignment did not really help my writing. I realized that I wrote my drafts less as a stream of conscious writing but more as a perfect paragraph. I wish I had changed how I did this assignment. I was actively editing while I wrote which left my draft and perfect paragraphs being quite similar. I do think this gave me the ability to work on my organization and be more aware of grammatical errors.