Please post your response to our last paper here. Remember to submit all of your posts to turnitin.com.
Gillespie Lab
Science, Education, & Technology
Please post your response to our last paper here. Remember to submit all of your posts to turnitin.com.
Gillespie Lab 
This paper focused on finding a way to categorize genes that can lead to cancer due to their mutations by looking at the gene expression of shRNAs. Overall, I found the paper itself difficult to read but the actual idea that the scientists were dealing with was very interesting. The paper was hard to read because I felt like it was hard to really understand the methods. It wasn’t until our in class discussion that it was clear that it was actually like two mini experiments by adding the short hairpin mRNA.
I really enjoyed seeing the different techniques that these scientists used in this experiment though because they were all techniques that we used in the lab. It made the paper, when it came to the figures, more relatable and also easier to understand. This paper used the concept of transformation by using the lentavirus to add the short hairpin mRNAs into the genome of the cells, PCR and microarray.
By using this new method to try and identify genes and the mutations in them, I think that it holds a lot of promises for the future of preventing cancer. By knowing the genes where mutations occur in different cancers, doctors would be able to inform patients and help to prevent them prior to cnacer. Of course with this technology, whole genome sequencing would also have to be used for the patients. Perhaps in the future whole genome sequencing will be available and this technology of identifying mutations and specific genes which deal will cancer will help prevent cancer
This paper talks about the use of the recently developed RNAi libraries. These new libraries have enabled systematic genetic studies in mammalian cells by using arrayed and pooled screens. This new information available gives scientist a starting point, from where to start researching for the complete mechanism that is involved with cancer. Once they understand the mechanism they can improve prevention, diagnosis and treatment. The RNAi libraries also allowed for the development of the shRNA; short hairpin RNAs (shRNA) that are lentivirally encoded RNAs. Using this shRNAs involves infecting cultured cells, and allowing the cells to proliferate for a given period of time. After the time has passed, the shRNAs are isolated using PCR amplification and measuring the abundance of these shRNAs; measuring shRNA abundance is done by microarray.
The idea of what the paper was trying to prove was very clear, but my short understanding of the scientific terms and the methods used really made it hard to fully understand its impact. From what I could understand there is a new type of experiment that can locate the genes expressed in cancerous cells. If perfected doctors may really be able to help recover from cancer, or to a greater extent being able to prevent it. This paper, like the other gene related disease papers, gave me a glimpse of what future technologies hold for the understanding of diseases and it makes me comfortable to know that someday us humans will not have to fear such deadly diseases.
“Highly parallel identification of essential genes
in cancer cells”
The article discusses the underlying genetic aspects to how cancerous cells continue to grow, multiply and thrive. Scientists are researching genes “that are essential for cancer cells with the proliferation and also altered in human cancers.” Then, hopefully after discovering the many genes responsible for cancer proliferation, they can further investigate the genes involved with the response of cancer cells in the body that allow them to thrive. By understanding these underlying causes and mechanisms, scientists can have a greater knowledge of cancer at the molecular level and possibly find more effective methods for treating the disease.
The study involved using RNAi mechanisms for understanding cancer at the genetic level. (RNA interface) In the study the scientists infected cells with small hairpin RNA (shRNA), then they isolated these cells and amplified them with PCR reactions. This enabled them to pin-point the alterations in shRNA abundance. They figured that they could use this method to figure out genes responsible for the proliferation of metastatic cells. The study was complex and a bit hard to understand if one does not understand all of the scientific terminology. But what I gathered from the study is that the purpose of their study was using methods in which they pooled shRNA and were strategically able to identify certain genes involved in the stages of progression of cancer cells.
Overall I felt the paper was difficult to understand but the methods and ideas the scientists used to relate certain genes to cancer was very interesting. By gaining a better understanding of cancer at the molecular level, we can better discover effective treatments.
In this paper, scientists discuss how the complete knowledge of genetic expressions underlying cancer will improve prevention and cancer treatment. They attempt to explain these cancerous mutations by analyzing shRNAs which are long strands that will cleave mRNA. Through out this paper, there are two questions that are being addressed- How can you tell someone their probability for developing certain cancers and if the same exact tumor is the wrong assumption would that form of therapy fail?
In order to begin the experiment, they developed a highly parallel pooled screening that employs a library of created by RNAi. In this screening, shRNAs cells will be infected so that they can proliferate, then isolate shRNA and measure the relativity. The scientists only want 30 percent to get infected because you want to ensure that one cell gets one virus. After the test, then you’ll get cells that have shRNAs that do not perform anything. It is good to use shRNA because it cannot be lost since its stuck in the genome.
A microarray analysis is also performed in which the glass slides are made up of complimentary sequence of shRNA. The enriched or depleted shRNA will represent genes that are affected by the variable used in this experiment. The FADD ligands will secrete on the broken cells and assemble disc complex. The apoptosis pathway will stop if one of the proteins are missing from the pathway. After this experiment was performed, scientists are now trying to build a library of genes that will assist cancer treatments.
Although it was an interesting topic, this paper was very difficult to read. There was terminology that was not easy to comprehend and the way the procedures for their experiment were performed was hard to follow. I believe that these scientists are working with great methods that could help prevent cancer. They are methods that could change the face of medicine as we know it and save millions of lives. There are several more studies that should be performed to ensure great treatment quality, but I believe this is a great step towards telling a patient their probability for developing cancer. Probabilities that could easily eliminate unnecessary, expensive tests that are currently being used to determine cancer. It is a technique that specify the necessary treatment that should be taken for a patient, rather than having them experiment with uncertainty.
The goal of this paper is to broaden our knowledge about cancer to enhance its prevention, diagnosis and treatment. The researchers in The Cancer Genome Atlas are using their efforts to identify the genomic mutations that are linked to each cancer type. In one cancer type, it has been proven that there are multiple different pathways to get there making it difficult to classify all of the pathways that get to the different tumor types.
From this fact alone I became discouraged because it sounds like an impossible task to discover all of this information from research. I began connecting all of the things we learned throughout the semester and thought about how maybe we have “personalized cancers” in a sense because we are all different and develop diseases differently. In the future, I do not know how they would discover the cure for cancer but at this point it seems nearly impossible.
Aside from that thought, there are most likely a limited amount of genes that are linked to the certain tumors essential for growth and related phenotypes in different cancer cells. So the method used in this experiment to identify the genes in 12 cancer cell lines is to ultimately perform a genome-scale pooled shRNA screen for positive and negative selection. Afterwards they also identified the genes linked t o the response of cancer cells to a certain treatment. It is important to find out responses to certain therapies to prevent any catastrophes like making the cancer more resistant to the next treatment if the therapy fails.
Scientists used their lentiviral RNAi library to assess cancer gene function and to incorporate “structural and functional approaches in the study of cancer.” In The RNAi Consortium library there were about 170,000 lentivirally encoded short hairpin RNAs (shRNAs) where 5 or more shRNAs target each of 17,200 human genes. They infected culture cells with a pool of shRNAs, let the cells reproduce, isolate their sequences from these cells through PCR method, and measured the abundance of shRNAs by cleaving the hairpins with a restriction enzyme and hybridizing them afterwards to a microarray complimentary to those sequences.
Lentiviruses are referred to as a retrovirus because the DNA sneaks into the nucleus and ends up in the genome. So you can never cure a cell of a retrovirus because the genome can never be altered. The host would be infected and gradually infect themselves without them knowing.
The shRNA is a hairpin shaped RNA that gets cut by an enzyme that finds an mRNA sequence to silences gene expression by destroying it. It is stable inside the cell and if you can carry the hairpin sequence in a lentivirus into the cell and into the genome. This makes the hairpin functioning and silences genes. The hairpins are stuck into the lentiviruses. These infected cells are sprinkled on cell cultures with“certain infectivity” or ratio of 30% infected cells out of the cell culture. The researchers only get one cell to get one virus.
Overall, the experiment shows which genes are responsible for certain cancer types from their over and under-expressions, find the data on how the treatments resulted for the tumors, and build the library of genes that line up with the treatment. This would inform us of the necessary and successful therapy, what is toxic, what tumor is affiliated, what can develop form this in the future, and what other tests should be performed.
Researchers in this article focus primarily on identifying the genes with important roles in cancer. Integrating both structural and functional approaches provided insight into therapeutic targets for treating cancer. The genome-scale high throughput methods are used for lentiviral RNAi library to systematically assess cancer gene function and to integrate structural and functional approaches in the study of cancer. 170,000 lentivirally encoded short hairpin RNA, with 5 or more independent shRNA targeting each of 16,000 mouse genes. And pooled screening approach involves infecting cultured cells with a pool of shRNAs, allowing the cells to proliferate for a period, isolating the shRNA sequences from the resulting cells by PCR and then using hybridizing technique by microarray to identify hairpins with no change, depleted, or enriched. Also the use of lentivirus to add the shRNA into the cell’s genome was an intriguing part of the experiment.
Genes that make the cell “oncogenic” were identified by immortalizing cell line specific genes. The results indicated that mutations in different cell lines are different. Cell lines was used a control to compare all the cell-lines which are not normal and is different. Likewise, the scientists were able to list the genes broken in the hierarchy of similar and different genes. Then, the genes regulated to show up in tumor were identified through the use of biomarker pathway which not only identified genes but a series of genes that may be targeted and the diagnostic approach to different cell tumor lines that look similar. Scientists identified “cell lineage-specific” essential genes, which exhibited a stronger phenotype in cell lines derived from a particular cancer type than in other cancer types.
This type of research relating to cancer therapeutics shows a lot of promise in developing novel strategies to help improve the patient’s ailing condition relating to cancer by providing an insight on the required therapy for particular cancer, the toxicity related to that treatment, pinpoint various techniques in the tumor progression and the probable cause for the specific types of cancer in future. These strategies will provide a great helping hand to treat cancer patients thus eradicating the disease eventually in future.
The goal of this week’s paper is to find genes and pathways that play a critical role in the formation and survival of cancer cells. The approach the researchers used is complicated and multi-faceted. The basis of their experiment lies in the use of short hairpin RNAs or shRNAs. They work similarly to siRNAs, by annealing to, cleaving, and ultimately “silencing “mRNA. With the use of shRNAs the researchers were able to perform microarray analysis for gene expression levels. Through their experiment, the researchers were successfully able to determine certain genes and how their lack of presence leads to pathways that cause cancer. The researchers suggest developing more shRNAs for each gene so that these types of studies can be expanded.
This was the most difficult and confusing paper to read this semester. Its technical language and lack of organization contributed to its difficulty. I know that the researchers are writing to a more educated audience so I don’t expect everything to be spoon fed. However, brief explanations of certain concepts and some organization (such as a methods, experiment, results, and conclusion section) would help the reader a lot in figuring the paper out. The shRNA method used in this experiment seems interesting but I wish they would’ve explained it better. The fact that it can insert itself with a virus and then cause the genome to make more hairpins is fascinating. The class discussion helped me understand a bit more what this paper was about, but it still makes it hard to put everything in this paper together.
Overall, I would’ve enjoyed this paper much more if it wasn’t so technical. I wish their goal and experimental procedure was better outlined.
The paper, “Highly parallel identification of essential genes in cancer cells” by Luo et al., focuses on functional characteristic of genes that would relate the certain cancer cells. Researching the cancer correlated genes and phenotype of the specific cancer, the scientists could find how the gene affects the immortality of the mutated cell. This research will contribute to approach diagnosis of cancer and help to use right therapy for certain cancers.
The researchers are using lentivirus that carries short hairpin RNA (shRNA) and injected into the cell. Lentivirus is one of kind of retrovirus which is that the virus does reversetranscription so that it ruined the genome sequence of normal cell-subsequently; it ruined the daughter cell and neighbor cells too by giving wrong genomic information. Especially, lentivirus could hold the shRNA and it is stable in the nucleus that the scientists easily watch what gene s is related to specific cancer by knock downing some gene expression. For the experiment, two groups are used that one was for control and the other was for the sample. On the sample cell culture, we focused a cell infected by certain shRNA and amplified the DNA of infected cell. After microarray the sample, we came out three outcomes which were same with the control, enriched, and depleted result. Any enriched and depleted genes mean genes that are similar or related to the variable that the scientists used in this experiment.
Generally, cancer is mutated cell that is not dying on time that it supposedly dies. So they used the apoptosis pathway. Apoptosis pathway is used for the cells to suicide themselves. Usually, the signal from ligand (FAS) turns on the sign that the cell should die. By injecting shRNA, the scientists could see what progress of the pathway is ruined and as a result what kind of certain cancer occurs comparing the phenotype of it.
I had no idea what the paper was talking about until I had class discussion. Still I had some confused concept of the experiment. Actuall, I doubt myself that I understand this paper right or not. The one truth is the researchers are smart that they could think about this experiment. They could use siRNA but they used shRNA, which more stable and easy to reach the inside of cell than siRNA that they could experiment steadily.
This paper talks about how different methods can be used in indentifying the genomic mutations associated with each cancer type. The paper introduced a type research with the use of RNAi libraries, which enable systematic genetic studies in mammalian cells by using arrayed and pooled screens. The screening approach involves infecting cultured cells with short hairpin RNAs (shRNAs) , which is created from the RNAi library and is a sequence of RNA that makes a tight hairpin turn that can be used to silence gene expression via RNA interference (RNAi). After we allow the cell to proliferate after the infection we use PCR to amplify the shRNAs sequences from the resulting cells and then measure they abundance in doing the microarray experiment.
This paper was very hard to understand and I had a really hard time trying to read through the part with the conducted experiments. But at the end it was interesting to learn about new technologies and new advances in the future that will help in discovering a cure and much sooner diagnose for this deadly illness. The other interesting part of the paper was the part that mentioned PCR and Microarray, which I have both done in lab so It made me feel kind of cool because I knew what they were talking about
Highly Parallel Identification of Genes in Cancer Cells talks about a systematic approach to identifying the structural and functional characteristics of cancer causing genes to specific mutations that controls cancer growth and tumor initiation. The important idea here is so we can make more accurate predictions about the types of cancer one is likely to develop. And hopefully we can come up with better treatments.
The method used is involved short hairpin RNAs or shRNA which mimic the function s of small interfering RNAs, by targeting specific region of the RNA and silence its function. shRNAs are linto viruses that gradually work their way into the genome of the host, eventually inhibiting. The goal of this experiment was to identify genes associated with cancer growth, more specifically proliferation. One pathway the apoptotic was chosen randomly, where shRNA infected genes were sprinkled were sprinkled with ligands to target apoptosis in a variety of cells. The result were that while some reacted with enriched functions others reacted with a decrease in function. FAS, FADD, CASP8, ARIDIA and CBX1 were the five regulators that are associated with cancer growth.
Furthermore, the experiment involved testing immortalizing cell line genes, which allowed them to generate hypotheses about cancer specific gene dependencies on a common type of cancer, chronic myelogenous leukemia (CML) which is represented by mutations in a single cell line the K562.
Overall I think this paper is extremely incredible because it provides so much insight into the field of pharmacogenetics. It goes to show that diseases in general can be linked to mutations in the genome, that as long as we continue to study the genome and crack down its intricate codes, we will be able to find answers.
Highly Parallel identification of essential genes in cancer cells.
This paper overviews the development and usage of Lent viral (slow virus) RNAi libraries to asses cancer gene function and to integrate approaches to the structural and functional aspects of the study of cancer. The library consists of hundreds of thousands of “short hairpin RNAs” or shRNAs, similar to the previously discussed siRNAs, they are able to cleave mRNA at specific points . The basic systematic approach is to infect a group of cells with shRNAs, allow them to proliferate cells, the harvest the genomic DNA, amplify the hairpin regions via PCR and the cleave the regions with and enxyme (for ecample XHO1) and the hybridize the remaining genetic material to a microarray slide. Upon lazer analysis of the mircroarray one will be able to identify which genes serve as biomarkers that may describe a mutation that leads to a specific cancer. For each cell line several infections were performed and measured the viral proliferation at various points in time( 3-4 days,2 weeks, 4weeks etc) to best asses the timeline of infection, growth, and survival of cells with different hairpin sequences.
Overall this method of research can improve healthcare in refining the treatment for patients. Knowing a patient’s genomic information and comparing it with the information harvested from such shRNA studies will avoid many costly and painful diagnostic tests.
The last paper was a primary research paper on a series of experiments that: 1) attempted to identify oncogenes that were essential to cancer sell survival and function, 2) attepted to identify the different pathways several cell types undergo as they bocome cancerous, and 3) attempt to gain an understanding of what each gene does and how the pathways work in order to potentially create a diagnostic for general, clinical, use.
The scientists started by creating a “library” of 17 200 “small hairpin” RNA -shRNA- placed into lentiviruses, a.k.a retro-viruses. These viruses were then exposed to 2 cell cultures that were put under 2 varying conditions: test conditions- where the cells were treated with either FAS, an antibody that “turns on” apoptosis in a normal cell, or etoposide- and reference conditions where the cells were allowed to proliferate on their own. Once the shRNA were incorporated into the cells genome, and the unaffected cells were killed off, the cell’s DNA was run through a PCR that amplified the hairpin regions and this sample was hybridized on a microarray to identify which genes were “enriched” od “depleted”. Those particular genes were then compiled onto a “heat map” that showed the abundance of particular shRNAs in particular cell lines. The mean was then taken across all of the cell lines to determine what is “normal” gene expression, and what’s not. From there the scientists complied a list of genes and cellular functions that must “break” in order for a cell to become cancerous.
In the end, the scientists basically verified the previous list of genes and metabolic pathways known to cause cancer. Being able to perform these simple lab tests ro find the links to the causes of cancer in specific cells however, indicates that it is possible to create a useful diagnostic tool doctors can use to identify a cancer line in a patient without having to put them through a bunch of costly, trial and error therapies.
This paper addresses an approach to enhance cancer treatment in humans. Researchers aimed to find relationships between genes/pathways and the formation/survival of cancer cells, they conducted this experiment with the help on a type of RNA called short hairpin RNA (shRNA). Short hairpin RNAs inhibit gene expression in mRNAs and lentiviruses are combined with the shRNA for purposes of completing the experiment.
One specific method used for testing was a screening process with the help of RNAi libraries cells could be studied. With the use arrayed, pooled screens and infected cells researchers infected cultured cells of shRNA so that they could proliferate, researchers were careful in maintaining the 1:1 cell to virus ratio. After proliferation researches used PCR to amplify the sequences from the cultures and then conducted a microarray experiment in order to measure the abundance. At the end of the experiment researchers were able to identify a few biomarkers and they saw that some cells had significantly more function than others. Researchers ended up concluding that their experiment was a success and that they are headed in the right direction for another breakthrough in cancer treatment.
This paper was pretty tedious to read, I most definitely did not grasp the concepts the first time around. But I believe the information being presented is very interesting and is obviously made for a more scientifically mature audience but after discussing certain terminology I was able to grasp most of the concepts. I believe that we are now even closer to the idea of personalized medicine and that a large breakthrough in the field therapeutics is not far from our reach.
The paper “Highly Parallel identification of essential genes in cancer cells” attempts to identify the different gens and mutations that lead to the development of cancer. The study is based on the concept that the function of genes that result in the characterisitc phenotypes associated in cancer is related to the structure of the genome. Both structure and function of genes are taken into account to develop new treatment and drugs for cancer patients.
It attempts to identify the genes associated with the continuous division of cancer cells so that new treatments and drugs that can block these genes may be developed. The RNAi library stores the gene functions along with its strucutral and functional association and relation to cancer. 17, 200 humna genes were targeted using short hair pins RNAs. Cultures were infected with these shRNAs, then amplified using PCR, and then hybridized on microaarays to determine expression.
The paper identified genes important to 12 cancers and using the data realted to these tumors scientists were able to conclude several oncogenes such as MYC and KRAS associated with cell differentiation in cancer cells. The pooled shRNA approach also allowed for the identification of genes that were associated with and interacteed with drugs, such as those involved in response to imatinib.
The researchers conclude that their method of approach is valid adn successful and that further studies using this approach will be beneficial and successful. However, more shRNAs are needed for each gene. Further research on different phenotypes associated with cancer adn their structural and functional analyses can help to identify more genes associated with cancer. This information can help us to develop drugs to interfere with cancer development.
This would be very useful for health care providers. If they knew which drugs works best for each different kind of cancer they could personalize each patients cancer treatment to get the best possible results and hopefully less sickness. This information can also be useful to drug companies who are trying to design new cancer drugs. If they knew the different pathways that cancer took and messed with they might have a better chance of stopping it. Knowing the genes and mutations that occur for certain cancers will let doctors be able to give there patients a different look at the possibility they have for developing certain cancers. They then might be able to give there patients different preventative care methods to reduce the risk of getting cancer.
This can also bring about a few problems as well though. First of all the doctors are going to be telling people how likely they are to get cancer and most likely the patients will want to start treatment right away to be sure the don’t get cancer but this can be completely unnecessary because there is always a chance that they never get it. Even if some is 99% likely to get cancer they could be in that 1% and end up never getting it. It is also a problem because you can actually do more damage treating someone with the wrong treatment then if you didn’t treat him or her at all. So if you treat them with the wrong drugs because you think you know they gene pathways you co do a lot of damage to your patient.
In this experiment shRNA or short hairpin RNA was looked at. shRNA silence gene expression through the same method as siRNA (cleaving mRNA). If someone has a certain endonuclease a hair pin RNA the loop part gets broken off and the 2 strands open and go into the genome and as an mRNA the hairpin gets put throughout the body and silences different things.
What they did was take the hairpins that are in the lenta virus that is in the infected virus and they sprinkle them onto cells. This is done with a certain infectivity how likely each cell in culture and the likelihood of one cell getting a virus (0.3 or 30% and 30% of the cells in the cultures get infected and 70% of the cells don’t get a virus). Many cells are used for so little virus to ensure that each cell only gets one virus. One should be more willing to lose a lot of cells because if a cell got 2 viruses it would mess up the results. After the cells get sprinkeled and then the test is done this test gets rid of cells that they don’t want. When done the cells are taken out, spun, and take the genome out of the cell. PCR is then done to amplify the hairpins and then it is run on the microarray. The spots are made up of complementary sequence to the shRNAs. Overall I enjoyed this paper, I think it went along nicely with what we were doing in class and was especially interesting because I am really interested in oncology.
The paper “Highly Parallel Identification of Essential Genes in Cancer Cells” aims to find better prevent, diagnose, and treat cancer through the identification of genes and different metabolic pathways and the mutations that occur that leads to the development of cancer. The information learned through these experiments would help create a database where drug therapies are given based on each affected individual’s genome. The RNAi Consortium’s library contains a collection of short hairpin RNA sequences that target 17,200 human genes. The shRNA is used to silence gene expression. The hair pin is a long strand of nucleotides that pair with the DNA. It pulls the complementary strands apart and uses it to destroy the RNA. The hair pin is stable inside the cell. The virus can be combined with the shRNA sequence and integrated into the genome of the cell to turn off specific genes.
The hairpins are combined with the lentiviruses. Each lentivirus only has one hairpin. They take put the hairpins onto a cell culture, however only 30% of the cells are affected. The small percentage of cells are not re-treated to ensure that each cell is exposed to only one of the hairpins. This way, researchers know that they are not studying cells that contain multiple hairpins and therefore misconstrues the results.
The experiment is run by putting the hairpins onto the cells. The control used would be treated the same as the experimental cell culture but leaving out the variable of the hairpins. The cells are spun down a tube, the genomic DNA is prepared, and PCR is performed to amplify the hairpins. These sequences are then cut with a restrictive enzyme, labeled, and allowed to run on a microarray for examination. The spots are made up of the complementary sequences to the shRNA, and from the spots, it can be determined which gene that it originated from.
The idea of the experiment seems simple enough but a problem that researchers must resolve is how the combination of genes together effect cancer development. Drug therapies could target specific pathways but it would be useless if the other pathways involved are not accounted for. Also, it would be important to note the sensitivity for each patient towards the drug that is being distributed.
This study tried to identify a limited number of gene pathways that lead to cancer. They stated that by recognizing both structural and functional characteristics of genes that cause cancer, will help identify and treat cancer.
To do this, researchers came up with a “pooled screening” strategy which works in the following manner: infecting cultured cell with shRNA (short hairpin RNA that silence gene expression by cleavage, similar to siRNA) and allowing the cells to multiply, followed by the isolation of shRNA (using PCR) and then examining the relative abundance of shRNA (depletion or enrichment, using microarray). The control used was just the media of cells but the experimental group contained media plus ligen which leads to cell apoptosis. The only way for cells to survive was to turn off some genes (silencing) and siRNA aided in breaking the pathway so the cell survived. The 12 cancer cells that they used showed substantial correlation.
The researchers also suggested some ideas that will further improve the study- increasing number of siRNA per gene, cost effectiveness, larger shRNA libraries. They concluded that feasible method can also be used for other studies not just proliferation of cancer cells and thus find a cure.
Unlike many of the other papers we have read in the past, this article presents its main focal points in the beginning of the paper.
Right off the bat, the reader is informed of the highly parallel correlation between the identification of essential genes and cancer cells.
A process that is further discussed in detail later on known as shRNA screening. To carry out this method, scientists have come up with a “pooled screening strategy” that involves infecting cultured cells with a “pool” of shRNAs (a close “relative of the siRNA), allowing for the cells to proliferate then measuring the relative abundance of the shRNAs (which are amplified y the process of PCR) through the process of microarray.
Through this process, scientists have identified twelve known oncogenes (ex. EGFR,KRAS,MYC, BCR-ABL, MYB, CRKL, CDK4) that are essential for the survival of cancer cells and are often altered in human cancers.
As the researchers have in this paper, I also believe that if this sort of identification can help identify the genes necessary for the formation of a certain type of cancer, then it could also be possible to discover the genes that participate in any other biological pathway.
In order to identify genes with important roles in cancer, a systematic approach integrating both structural and functional methods will help assess genes for their contribution to the development of cancer and it’s phenotypes which is complementary to the structural classification of the cancer genome. To approach this task, RNAi libraries targeting human and mouse genomes were utilized for the application of a high-throughput genome scale method in order to systematically assess cancer gene function. To apply RNA in a genome study, a highly parallel “pooled screening” strategy using the TRC (The RNAi Consortium) library of about 170,000 lentivirally encoded short hairpin RNAs (shRNAs), with 5 or more independent shRNAs targeting each of 17,200 human genes as well as 16,000 mouse genes were collected. The process required infecting cultured cells with a pool of shRNAs and allow proliferation time for cells, then the resulting cells were isolated from the shRNA sequences by PCR amplification to measure the relative abundance of the shRNAs. This was done by cleaving the hairpins with an enzyme and hybridizing them to a microarray complementary to the shRNA sequences. In the experiments that followed, a smaller library containing 45,000 shRNAs was used. This 45k shRNA pool corresponded to ≈9,500 human genes. In the initial test, 2 positive-selection screens were performed, in the first screen Jurkat T cells were infected with the 45k shRNA pool, each cell receiving 0.3 shRNAs and ≈200 independent cells in order to identify genes which rendered T cells resistant to apoptosis by FAS activation through its inhibition. After the remaining cells were isolated from the uninfected cells, they were treated with a dose of anti-FAS antibody sufficient to rid the number of infected cells. To identify resistant shRNAs, shRNA overrepresntation in the surviving treated cells were measured. To filter out off-target or ineffective shRNAs, genes were defined as “hits” if 2 independent shRNAs were ranked in the top 0.9% of overrepresented shRNAs. The results indicated 11 hits, out of which 9 were confirmed. Reliable gene expression levels were measured for 7 of these genes and 5 were found to show strong correlation between the level of resistance to FAS-induced apoptosis and the level of gene knockdown. This confirmed that the shRNA effects were indeed on-target. 3 out of the 5 genes had well-established roles in FAS-induced apoptosis (FAS, FADD, and CASP8) and 2 previously undescribed genes (ARID1A, a SWI/SNF chromatin remodeling complex component, and CBX1, a chromatin silencing protein). The 5 effective shRNAs for the 5 genes also inhibited FAS-induced CASPASE 8 cleavage, suggesting that, the 2 undescribed genes like the 3 known genes act upstream of CASPASE 8 activation. Missing downstream apoptosis genes could be an indication of false negative results or a finding that reflects unneeded downstream genes for activation of apoptosis due to functional responses or compensatory processes. The second positive selection tried to find genes whose inhibition rendered H82 small-cell lung cancer cells resistant to etoposide, a drug used to treat small-cell lung and other cancers. A high dose of etoposide was used to eliminate H82 cells, and a suppressor gene emerged: TOPOIIA. It was shown that reduced TOPOIIA expression resulted in etoposide resistance in SCLC lines. Besides these two positive screen tests, negative-selection screens were used to identify genes that are crucial for the proliferation of specific cancer types, which involved the infection of cell lines with a pool of shRNAs and identification of underrespresented surviving shRNAsm similar to the positive-selection screens. For the negative screens, RNAi screens with 45k shRNA pool in 12 cancer cell lines were performed. These included small-cell lung cancer (H82, H187), non-small-cell lung cancer (A549, H1650, H1975, HCC827), glioblastoma (LN229, U251), CML (K562), and lymphocytic leukemia (Jurkat, SUPT1, REH). 5.4 million measurements were recorded for the abundance of the 45,000 shRNAs in conjunction with the 12 cell lines and 10 replicates.
“Highly Parallel Identification of Essential Genes in Cancer Cells talked about goals to prevent, diagnose, and treat cancer through the detection of oncogenes and mutations that lead to the development of cancer. One method used for experimentation was a process that studied RNAi libraries cells. In The RNAi library there were about 170,000 encoded shRNAs where 5 or more targeted each of human genes. In this screening, shRNAs cells would be infected so that they can grow, then scientists would isolate the shRNA and measure the relativity. The scientists only want a certain percentage to get infected to make sure each cell gets a virus.
A microarray analysis is then performed. The slides are made of the ShRNA complementary strands. The results were the same as the control, enriched, and depleted. There were five gene regulators in the ocogenes that were associated with cancer growth, the FAS, FADD, CASP8, CBX1, and ARIDIA.
This paper was very hard to understand as most of the other papers. I still had a few questions after I finished reading the paper such as will this help scientists create a new medication for help fight cancer? and to what accuracy do the ocogenes have in developing cancer in humans without environmental effects?
The topic of this paper was very intriguing to me. Even though I did not understand the paper at first, the discussion helped me bring together the interesting parts of the paper. The use of shRNA’s to pinpoint certain genes not only helps in cancer treatment, it also could be used in other health care related areas. The integration of the shRNA’s into lentivirus’ took advantage of the virus’ slow rate of infection. The 30% of cells that were infected were left alone to proliferate and were then put under the PCR process. The shRNA’s were amplified by introducing a splicing enzyme that allowed the shRNA’s to anneal to cDNA on a microarray. The analysis of infected test cells, with the use of micro array analysis, helped scientists identify certain cancer cells that had amplified expressions compared to the control cell (non-cancer cell). The creation of a library would help future diagnosis of patients and that specific treatments would be created for individuals.
This paper focuses on improving knowledge about molecular mechanisms underlying cancer so that it will be easier and more efficient to help cancer patients. This is done by what the scientists call “systematically characterizing the structural basis of cancer”. This is basically figuring out the functional basis of cancer and simply; identifying the genes related to cancer growth and related phenotypes. This systematic approach was done by using RNAi libraries to target human and mouse genomes for the application of genome – scale method. To apply this RNA interference at a genome scale, parallel “pooled screening” was developed using the TRC (the RNAi Consortium) library that contains approximately 170,000 short hairpin RNAs (shRNAs) with 5 or more independent shRNAs targeting human genes as well as mouse genes. This screening is done by infecting cultured cells with a pool of shRNAs, so that the cells will proliferate then isolating the shRNA sequences by PCR amplification from resulting cells. Then the shRNAs were measured by cleaving the hairpins with enzymes to hybridize them in a microarray slide. The procedure is simply infecting cultured cells with shRNA which are short hairpin like RNAs that silence gene expression by cleavage. Then the PCR was used to multiply cells and to isolate shRNAs followed by measuring the abundance of shRNA by microarray.
The experimental just explained in this experiment is done the same way as the control but only with the shRNAs. The Control group is performed without the hairpins. After the microarray is run, the spots are made of the complementary sequences to the shRNA and the visualization of the spots will help determine which gene that it came from. The experiment is quite straightforward, but the technicalities make the experiment sound more difficult than it actually is. This systematic characterization of genes related to cancer can help identify potential drug targets but it doesn’t account for multiple pathways of genes. Genes very rarely work alone, therefore the question is what pathways, and what genes work together to commit growth of cancer? If more and more cancer genes can be identified, then hopefully this task of figuring out the blueprint of cancer pathways can really find a solution to drug therapy.