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result and conclusion

Submitted by ziweiwang on Thu, 11/21/2019 - 18:44

The efficiency of the mutated RHO allele cleavage is 70.7% whereas the efficiency of the wt RHO allele is 3.8%. In the wt there was 8% insertion and deletion using this method but in the mut, there was 93% insertion-deletion in this method. In the wt, the rho localized on the plasma membrane while the P23H mutant protein remained trapped in the cytoplasm. The two insertion-deletion mutations the antibodies were not seen in both of those mutations. In this experiment, after one month there was a higher thickness of OS was higher in the CAS9-VQR and sgRNA mut compared to cas9-vqr treatment only. There was no change in the ONL thickness did not differ depending on the treatment. At three months, there was a slight difference in ONL thickness, with cas9-VQR and sgRNAmut having thicker ONL layer and cas9-vqr having the least thickness. The effect on the thickness of OS layer remained the same. The CRISPR-VOR the uninjected eye presented better response compared to the treated eye, indicating that there is a detrimental effect that the procedure has on the function of the retina.The injection method allowed for the gene delivery. All subtypes of the retinal cell types were targeted. However, the rod photoreceptor had the highest transduction and the glial cells had the lowest transduction. GFP transduction was robust and diffusion the ONL. The RHO mut gene presented a significant level of cleavage GFP high cells exhibited a higher accumulation of indels High effect in unedited P23H allele. Every delivery system that was compared had the same amount of frameshift mutation in the allele. If the mutation resulted in the stop codon, it was more likely to disrupt the expression. The GFP plus cells had a 3.2% efficiency while te GFP high mutation had a 48% efficiency. 

goal and method

Submitted by ziweiwang on Thu, 11/21/2019 - 18:42

The goal of the research is to validate a CRISPR/CAS9 inactivation treatment for the P23H mutant for Renintitis pigments and put the treatment to test in vivo using the AAV delivery system Experiment 1 that was shown in the study tested whether RHOgene allele-specific targeting could be achieved in vivo. To do this, the researchers attempted to deliver the CRISPER CAS9 VQR system components in the retina of mice by invivo electroporation. To do this, the researchers devised a vector that pCAG vector was electroporated with GFP reporter in the RHO Het eyes.  The invivo efficiency was assessed by harvesting the eyes and purified by FACS sorting and molecular analysis was done.  The mutation frequency of the wt rho gene was also analyzed to test the effect of treatment on Wt cells. From experiment 1, it has been noted that the P23H mutation had high rates of frameshift mutation which leads to the inactivation of RHO allele. The mutant allele is not effective in treating the disease. To asses, this problem The RHO gene mutations that occurred the most often were overexpressed in mice P19 cell line, and colocalization of the Rho n terminus antibody and Rho c terminus antibody.  because the experiment 1 and two resulted in an efficient delivery of CRISPR/CAS9 elements in the retina, with targeting rate of the mutant gene, the researchers attempted to asses the photoreceptor degeneration in mice that was treated through an histological analysis to see what the result of the treatment in the degeneration of the photoreceptor cell. The histological study was done at 1 month.  The same histological study was done again at 3months. To test the injection method of the treatment, the intravitreal injection of AAV2 variant was given, and the histological studies of the retina were performed for the expression of transgenes in the tissue as opposed to the retina specifically. The AAV-PHP mediated targeting of mutation of the whole retina was used with CRISPER/CAS9 was used and the two different approaches are both used, and the efficiency of treatment was assessed for the genetic change. In this experiment, the possibility of enriching for the editing events using GFP transduction using FACS sorting. Using the data that is observed in experiment 6, the researches subjected the High GFP and low GFP cells to NGS analysis to refine the indel analysis. To do this, a PCR of the RHO allele was done to asses their incidence and the editing frequency in retinal samples. This experiment aimed to confirm the selectivity of the approach for the P24H RHO allele.

paper summary

Submitted by ziweiwang on Thu, 11/14/2019 - 23:51

In this research paper, the research shows that the disease in the photoreceptor cells also affects the cells that are around those cells, this changes how the disease should be studied. In the study, the conclusion of the experiment is that the loss of photoreceptors changed the number and morphology of the glial cells such as astrocytes and Muller cells. The change in the number and morphology of the glial cells affected the retina and as a result, may be an important part of the retinitis pigmentosa which was previously only thought to affect cone cells. Because the disease affects other cells and not just the cone cells, there is a need in the cellular and tissue level model to mimic how the cells behave in the disease. In addition, any further research into the treatment for the disease needs to take into account how the treatment would affect the cells surrounding the photoreceptors. For example, if a drug only really works to fix the one problem that is wrong with the mutated protein, this may not necessarily fix the damage that has already present in the surrounding tissue. By taking into account these issues, the treatment will have a higher chance of performing well in the clinical trials, leading to a faster treatment that can be released into the market.

reflection on learning biology

Submitted by ziweiwang on Thu, 11/14/2019 - 23:23

It kind of occurred to me that when writing for this assignment, I often write from the assignment that was assigned and the techniques and how to work in a lab, and almost no information about different biological phenomenons. When I do, it's usually from other studies and reviews. I think that one of the reasons why I'm so interested in papers and techniques that would be used in the lab and not the biological processes because I think that the biological discoveries are so fluid. While that fluidity is nothing compared to something like, say, sociological science, it is also true that a biological finding may be wrong or contain flaws. I've always had an almost unhealthy interest in discovering the flaws of the study and looking at it, from the first epidemiology class that I took on the cancer epidemiology where I read papers and discussed them with the people in the class. I think that this carries over to my biology undergraduate degree as well because I'm planning to take 3 journal club courses. It's further interesting that I chose these because I'm not good at reading papers. But it's important. I've been taught how to read papers since I was in introductory biology in high school, but I still have trouble reading them, especially since they often come from disparate fields. I want to learn how to read scientific papers because I'm not good at it, and the university does not offer classes on how to read papers. This and how to work in a lab is something that I think should be taught as much as writing because those three are all of the things that are going to be needed in the future. While things like advanced courses like immunology and neurobiology is important, with the abundance of information in the form of books and google, I want to prioritize skills that I would think I regret not learning, and for me, those would be learning how to write well, read papers and learning integrate myself into a lab and conduct research. 

southern blot

Submitted by ziweiwang on Thu, 11/14/2019 - 21:56

One of the most well-used techniques in the lab is the southern blot. This method has been in use for a long time and continues to be relevant within the field of biology. not an easy feat considering how fast the field is moving, and how new techniques are quickly replacing the old ones due to their ease, cost efficiency and decrease in time. However, southern blot has still been relevant enough so that in the job description previous experiences with southern blot have been listed as a requirement. To do this technique, the first thing that needs to be done is to do the last step of genotyping: gel electrophoresis. by running the gel, The different RNA fragments are separated. The RNA is then transferred onto a membrane. Then the membrane is then left in a container that contains a mix of proteins. the resulting mixture is then left to shake allowing the different proteins to interact with the segments of the genetic material. After some time, the membrane is then exposed to UV light to see what binding happened. The band indicates the interaction that happens between the genetic material and the protein/molecule of interest. Because mRNA is used, the southern blot can tell the gene expression of the cell. Similar things can be also done using protein, and DNA. another technique that is similar is DNAase analysis where the protein bind to DNA before the gene is digested and ran on a gel. Because the sizes and the places where the DNA is cut, there is a ladder running down the entire gel. However, because the protein are bound to the protein cannot be digested, there is a blank spot where it indicates the spot where the protein binds to the gene. 

summary of the study

Submitted by ziweiwang on Thu, 11/14/2019 - 21:29

The basic aim of the paper is to analyze the effect of thyroid level on OPCs marker and MVP level in SVZ, olfactory bulb, and optic nerve. The way that this was done is by increasing or decreasing the thyroid hormone by giving PTU or T3 thyroid hormones. The result of the study is that hyperthyroidism caused a decrease in MBP and a decrease in differentiation while hyperthyroidism caused an increase in MBP and an increase in the maturation of oligodendrocyte. From this result, the most reasonable way to infer how the result of this study can help me infer how the T3 and T4 hormones affect the neural stem cell is that there is an increase when there is a decrease in thyroid level, and a decrease when the thyroid level increases. The reason why I think this is because when the neural stem cells divide, they divide into two, and then one of the resulting cells would differentiate unevenly leaving one of the cells unusable and the other one would not be a stem cell Because of this, there would be an increase in neural stem cell because the decrease in the thyroid hormones I think would stop the division at when the neural stem cells are dividing evenly and as a result, there would be an increase in the stem cells. The increase of thyroid hormone would cause the cells to split unevenly and as a result, there would be fewer neural stem cells.

conclusion and the impact of the study

Submitted by ziweiwang on Thu, 11/14/2019 - 20:57

The conclusion of the experiment is that the loss of photoreceptors changed the number and morphology of the glial cells such as astrocytes and Muller cells. The change in the number and morphology of the glial cells affected the retina and as a result, may be an important part of the disease which was previously only thought to affect cone cells. 

    This research shows that the disease in the photoreceptor cells also affect the cells that are around those cells, and as a result, there is a need to take into account for future research for cures and treatment that there is a need to also look at the cells around the main cells that are affected so that the normal quantity  and the morphology of the cells can be restored. In addition, one of the problems that were in the study is that the study was done on transgenic mice, where the gene with the mutation was inserted into the gene, causing it to have three copies of the gene. a more accurate way of making the model would be to make that mutation directly in the genome without increasing the gene number.


resultof the study

Submitted by ziweiwang on Thu, 11/14/2019 - 20:56

In the first experiment, the astrocyte morphology and distribution were observed. As shown in figure 1, there were significant increase in astrocyte cell number in the P23H rate compared to the control rates in all retinal regions. In addition, P23H rats were associated with hypertrophy, of astrocytes which also saw an increased GFAP immunoreactivity (figure 2,3). In P23H rats, there also showed a disrupted vessel that was significantly regressed. There is the formation of blood vessel tangles at 12 and at 18 months. (figure 4). 

In the second experiment, the result is that there is a relationship between the Cx43 and the hyperplasia and hypertrophy of the P23H astrocytes (figure5). There were also higher expression fold charges in the P23H compared to the control. In the experiment that indicates the tissue stress, P23H the diseased retinas showed GFAP immunoactivity in the muller cells, indicating that the tissue is under significant stress and that photoreceptors were significantly reduced in the mutant rat compared to the control (figure 6,7)l. In addition, from the staining it can be seen that the glial component of the retina expanded, filling the space that was left by the photoreceptors. In the experiment, it has been shown that in the mutant cells, the muller cells were hypertrophied, assumed to be due to the retina. There were also altered the distribution and orientation of cone cells(figure9). In general, the GFAP labeling indicated early retinal gliosis. The number of astrocytes was also higher in the mutant cells as compared to the controls. The astrocytes also degenerated as the rats aged.



goal and method of the study

Submitted by ziweiwang on Thu, 11/14/2019 - 20:55

    The goal of the research was to investigate the microglial changes during retinal degeneration in P23H mice. Specifically, the study tries to show that the degeneration of photoreceptors affects the cells around those cells.

26  P23H rats were used in the study along with 26 control rats. This would enable the researchers to study the 

    Protein expression was analyzed using immunoblotting. The protein was run on a membrane then the membrane was probed using 43 different antibodies The detection were then performed. The immunoblotting analysis would indicate the amount and the level of different proteins that are available in the retina. The result of the immunoblotting are shown in figure 5, 7

    The mice were euthanized and the eyes were enucleated and fixed in paraformaldehyde for one hour and cryoprotected in sucrose. The eyes were then dissected and the eyecups were processed and frozen. Then the sections were cut and mounted on a slide. This will allow for the image processing of the retina of both the P23H mice and the control mice, allowing for comparison. Then Retinal immunofluorescence was done on the mice, using the same antibodies and lectins. The previous sections were immunostained using the GFAP antibody, Gt antibody, Cx43 antibody, GS-IB4, and GAPDH antibody. These antibodies stain certain proteins in the retina and as a result, allow for a visualization of the slide that is obtained previously. The slides that were stained with the antibodies were analyzed for astrocytes the GFAPand TO-PRO-3 was used to identify astrocytes while the lectin was used to measure blood vessels astrocytes were counted manually, which allow for the examination of how much astrocytes there were in the retina. The result of histology are shown in figure 1,2,3,4,5,6,8,9.


background pharagraph 1

Submitted by ziweiwang on Thu, 11/07/2019 - 23:23

Hox genes are a family of genes that are vital to an animal’s embryonic development. The HOX genes belong to the homeobox gene family, which is the second-largest gene family. The Homeobox genes codes for the homeobox domain which are 61 amino acid that forms a helix turn helix structure. Homeobox domain is present in transcription factors that are important in development. Mutations in either the homeobox domain or in the HOX gene can cause serious developmental diseases. The expression pattern of HOX genes dictates the body plans of animals. In vertebrates, HOX genes are organized into clusters that are on different chromosomes named abcd which each contain 9 to 11 hox genes. In this proposal, we are focusing on HOXC genes. There are 9 HOXC genes. They are named HOXC4, HOXC5, HOXC6, HOXC8, HOXC9, HOXC10, HOXC11, HOXC12, and HOXC13. All of the HOXC genes reside on chromosome 12.


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