ziweiwang's blog

The goal of this experiment is to determine the characteristics of the unknown mutants. 

In this experiment, Yeast cells with unknown mutation that was made using UV light was crossed with other unknown mutant cells and other known mutants in order to characterize the unknown mutant cells. In this experiment, I crossed the unknown mutant colonies with known mutant colonies on an YED plate and then replicate the colonies on a MV plate and a MV+adenine plate. Using the unique characteristics of the different plates and the understanding of complementation, I analyzed the data to characterize the unknown mutants. 

RESULTS

    In the experiment that I conducted, in order to know the genotypes of the unknown haploid yeast cells, the unknown yeast cells were crossed with known mutant yeast cells used in previous experiments and with each other on an YED plate. Then the resulting plate was copied onto a MV plate and an MV+adenine plate. In general, the control was expected and there some crosses such as unknown alpha1 x ade 2a that produced a visible mutant, but the majority of the crosses resulted in complementation and as a result did not have a visible phenotype that could be seen in the YED plate. However, there were some mutation that was seen when a seemingly normal colony was transferred into an MV or MV+adenine plate.

These articles are about using bioinformatic in order to identify targets for drugs and drug resistance using data that was previously obtained and stored in the databases. 

Musa A, Ghoraie LSoltan, Zhang S-D, Galzko G, Yli-Harja O, Dehmer M, Haibe-Kains B, Emmert-Streib F. A review of connectivity map and computational approaches in pharmacogenomics. Briefings in Bioinformatics [Internet]. 2017 :bbw112. Available from: https://academic.oup.com/bib/article-lookup/doi/10.1093/bib/bbw11

In the YED plate, there were two red diploid colonies and one pink colony (figure 1). The red colony was from unknown alpha 1 x ade2 a and unknown alpha 1 x unknown alpha 1. The pink colonies were from the cross between unknown alpha 1  and ade 1a. In the MV plate, the result was the exact same copy of those that was found inYED plate (figure 2). In the MV plate that contain adenine, all of the colonies were white (figure 3). The exact shade of the plates can be seen in the figure. The result of the study was that most of the colonies on the plate were white this indicates that im majority of the cases, the complementation has occurred.

In the MV and YED plate, there were many white colonies. The reason for the white colonies in the MV plates is very likely due to complementation. In the YED plates, it is possible that some of the white colonies have a mutation in ade2. However, because the adenine that is required to grow a colony of ade2 mutants is lacking in the MV plates, it is unable to grow. If a colony is growing on the MV plate it can be considered that the  Because the complementation only occurs if the mutations were on the same gene, this indicates that if the resulting diploid colony was white they do not have the mutation at the same gene. This indicates that unknown alpha 2 and the two unknown a do not have mutation at either ade1 or ade 2 since the resulting diploid cells white when crossed with both ade 1 and ade 2.

Experiment 1: Neuromuscular SHIRPA

The scores increased significantly from week 3 to week 24, indicating a decrease in muscle usage. The adult C22 mice were the most affected and C3-PMP mice were intermediate. The things that were most noticeable were in tremor, gait and tail elevation and escape strength. (Figure 1)

Experiment 2: Electrophysiology. 

    The mean NCV was lower in C3-PMP mice and in C22 mice. Than in wt mice. C22 mice had a lower amplitude compared to C3-PMP mice and wt. The mean CNAP was decreased significantly from 3 to 48 weeks in C22 mice. But not in C3-PMP mice. (table 1)

Experiment 3: Histology and morphology

    In the C3 and CC22 and had numerous inappropriately thinly myelinated and unmyelinated fibers at 3 weeks compared to the control. The number of normally myelinated fibers increased in C3 and C22. There were more inappropriately myelinated in the motor branch compared to the peroneal nerve (Figure 3). Many amyelinated and thinly myelinated fibers and small diameter fibers that were hyper myelinated were found in both strains. There were occasional atrophic fibers and loss of Schwann cells at all ages of the C3 and C22 mice. All abnormalities were more frequent in C22 mice compared to the C3 PMP mice (figure 4). Macrophages were seen in C22 but not in C3 mice, which implies axonal damage. There were no regenerative fibers not identified with either the CC22 or C33 model(figure 3). 

Putting it all together:

    In adults, a higher neuromuscular SHIRPA score was associated with a lower number of fibers and lower CMAP amplitudes. Lower MNCV is associated with higher SHIRPA scores and lower number of axons. (figure 5)

Prep work: The mice were first genotyped to confirm mice’s genotype. The mice that were used for this experiment were created using cc22 mice in a C57BL/6J x CBA/Ca background, which have 7 copies of the human PMP22 gene. The CC22 mice were then backcrossed with wild type for ten generations. The researchers then isolated mice with milder phenotype and reduced PMP22 compared to the CC22 mice (3 or 4 copies instead of 7), and had a more stable genotype. All 3 types, The mild phenotype, C3-PMP, wt, and C22 were used for the experiment.

Experiment 1: neuromuscular SHIRPA

    In this experiment, the mice were assessed for neuromuscular function using the SHIRPA protocol. SHIRPA protocol contains 19 items to check for, such as body tremors and spontaneous activity as well as tail elevation. Using the items, the mice were each given a score from 0 to 43. 

Experiment 2: Electrophysiology

    In this experiment, mice were anesthetized and the sciatic nerve and caudal nerves were studied on an EMG machine to study motor conductivity. The electrodes were placed at the medial ankle and at the sciatic notch. Wave pulses were delivered and CMAP amplitudes were recorded the MNCVs were calculated. The needle electrode was inserted again to study the caudal nerve and CNAP and NCV were recorded

Experiment 3: Histology and Morphology

.     Mice were deeply anesthetized, blood was flushed shout and the body was preserved. Then the peroneal, femoral, and lumbar nerves were physically removed and fixed in formaldehyde overnight.  The samples were then processed into the resin, and light and electron microscopy were performed on the samples. The samples were stained using thionine and acridine orange for light microscopy and stained with methanolic uranyl acetate and lead citrate for electron microscopy. Morphology was performed by fitting the nerve to the stage and cross-section of the nerve were analyzed. 

Post experiment: Statistics. 

The data was gathered from the experiment were analyzed using descriptive statistics. The differences between the different mice were evaluated using the unpaired t-test.

The goal of the research was to study the pathology of myelin and axon in PMP22 overexpressing mice, a mouse model often used for CMT1A. The research question that is asked in the research is what is the change to the mice’s muscle tone, electrophysiology, and histology in the course of 1.5 years?

The experiment showed that the phenotype that is measured through noninvasive measures is indicative of certain morphology of the cells in mice and that the disease gets worse both in behavior and in morphology, histology, and pathology as the mice age. In addition, the C3 mice, in general, had less severe symptoms compared to the C22 mice but had symptoms whereas the control did not have any symptoms. In general, the C3 mice would be the better model for CMT1A.

The impact of this research on the disease is the creation of new model of CMT1A mice, which have a relatively more mild symptoms, and as a result would be more effective model compared to C22 model. This is important because the severity of C22 mice means that the treatment that work in C22 model would less likely be able to work in humans because humans generally do not have symptoms that severe. By having a less severe model, it will allow better replication in humans, saving time and money. 

Genotyping is a common technique learned in the genetic lab used to figure out the genotype of animals form their DNA. A proper genotyping is done in three distinct steps. The first is DNA extraction, the second is PCR, and the third is gel electrophoresis. The first step is DNA extraction. This is typically done using silicon columns. In this method, the lysis buffer and proteinase k is added to the tissue and incubated. The tube is then spun so that the supernatant can be removed without contamination. When this is done, the supernatant is then put into a spin column. It is then spun. This allows for the DNA to bind to the silicon and the rest of the supernatant to be separated from the DNA. The column is then washed using a wash buffer to remove any impurities and the column is then put into a new collection tube and eluted. The eluted solution contains DNA. The DNA is then put into another tube, and buffer, forward primer, reverse primer, MgCl2, and taq Polymerase are added. This is then heated and cooled at the optimal temperature for the gene, allowing for the duplication of the desired DNA. The resulting solution is then put into a well of the gel, and the gel is then ran using electricity. The resulting gel is able to show whether the gene is present or not. However, every gene is different in how it shows in the gel. One of the things that can be done if there is a relatively large amount of soft tissue is to skip on DNA extraction and just boil the tissue at 95 degrees in NaOH, adding tris HCl after. This is a more crude way of extracting DNA however, it is effective in some tissues, such as mouse ear, and often saves time. 

Genotyping is a common technique learned in the genetic lab used to figure out the genotype of animals form their DNA. a proper genotyping done in three distinct steps. The first is DNA extraction, the second is PCR, and the third is gel electrophoresis. The first step is DNA extraction this is typically done using silicon columns. In this method, the lysis buffer and proteinase k is added to the tissue and incubated. After this is done the tube is spun so that the supernatant can be removed without contamination. When this is done the supernatant is then put into a spin column. It is then spun. This allows for the DNA to bind to the silicon and the rest of the supernatant to be separated from the DNA. the column is then washed using wash buffer and the column is then put into a new collection tube and eluted. The eluted solution contains DNA. the DNA is then put into another tube, and buffer, forward primer, reverse primer, MgCl2, and taq PCR are added. This is then heated and cooled at the optimal temperature for the gene. The resulting solution is then put into a well of the gel, and the gel is ran. The resulting gel is able to show whether the gene is present or not. However, every gene is different in how it shows in the gel. One of the things that can be done if there is a relatively large amount of soft tissue is to skip on DNA extraction and just boil the tissue at 95 degrees in NaOH, adding tris HCl after. this is a rougher way of extracting DNA however, it is effective in some tissues, such as mouse ear. 

3d Fluorescence imaging is accomplished similar to how flow cytometry works. Both of them rely on tags that have fluorescence attached to them, are used most often in molecular biology in order to determine what enzyme is interacting with which ligand. The two things that are being looked at are both tagged in different colors. The tags are then scanned for with a laser to produce the raw data. The data is then processed so that the information can be processed. This is accomplished by establishing how close the two tags are. It is assumed that if the tag that has the molecules attached are close to each other, then it would make sense that the two are interacting in a chemical way. However, this would depend heavily on the quality of the imaging because the precision of the data is wholly reliant on the quality of the image. In addition, this also has a problem of never actually proving that they are interacting. They can appear to interact but never actually interact. In addition, the coefficient that is obtained from the result is heavily dependent on which one it is based on. For example, if there were a large amount of the ligand, and small of the receptor, the coefficient of ligand to receptor would be much smaller compared to receptor to ligand.  The positive of the technique is that it is relatively cost-efficient, and provide quantitative data from the image. However, this technique is als extremely labor-intensive and can only work well in a few experiments.