The poor girl Essay Example

The poor girl Paper Then I realised my own mum saw her just two weeks ago and she was also responsible for her death and could have helped her. The thing that bothered me was that she didnt even really even care that much; she was that kind of cold hearted person. Anyway it turned out while working at the committee that she was prejudiced against her case and did not help her. Mum hardly cared, I was quite shaken and upset and that was before I heard that she was pregnant; and thats not all mum knew it! Mum also shared her views with other people in the committee so that there was no one there to help the poor girl. I still cant forget the words: go look for the farther of the child it is his responsibility. It was so terrible I was trying to get over something and then another thing started! I would never look at my mum the same way as I used to; I knew that she was cold hearted but not to that extent. There was more to it though, she said that she werent responsible and said that the girl told us that the father was the one mainly in charge and that she would not marry him because he was a young, silly, wild and drank too much. For a moment I was a little bit more relieved knowing that it wasnt all her fault as I previously did. We will write a custom essay sample on The poor girl specifically for you for only $16.38 $13.9/page Order now We will write a custom essay sample on The poor girl specifically for you FOR ONLY $16.38 $13.9/page Hire Writer We will write a custom essay sample on The poor girl specifically for you FOR ONLY $16.38 $13.9/page Hire Writer Daisy was also surprisingly enough to me getting money from her but not excepting it because it was stolen! I was quite contented; she would rather live penniless then have stolen money. Mum then said that the father was a drunken, young idler thats all the more reason why she could not escape and rounded it off by saying if the death is anyones fault it is his. I felt that everything was going wrong for the whole family and then thought of Eric and later realised that he was the one, he was in charge; it just suddenly sprung into my mind. I was so distressed and then tried to tell mum what I had just realised. It was so awful trying to tell mum who it was especially after all the stuff she had been saying, she found it really hard to believe but then accepted it. Later on Eric came in all pale and distressed, he understood what we all found out about him and we all just looked at him with our inquiring stares. It was quite a sad moment we were angry with him, shocked and in a way I felt a little bit sorry for him. The inspector started to ask Eric some questions. After mum heard what Eric had to say she acknowledged the fact that all of it was true. She was about to break down so I had to take her out of the room. But later she just came back in curious to know what was going on. Apparently we were told by dad that Eric had admitted he was responsible and had stolen money from the office, fifty pounds! He said that he was meant to pay it back but I was not sure whether to believe him or not. A lot more dreadful things would happen later, it turned out that there was a big argument between mum and Eric I was feeling scared and wanted it to stop Eric was blaming everything on mum. He was saying the most horrid things like mum killed her and mum killed her own grand child; Erics son. The whole thing just came to an end with the words of the inspector: stop he said in such a compelling way that everybody did. Finally it came to an end he said he has found everything he needed to know and said that each and everyone of you were in charge of helping the girl die and not only that but he told us never to forget it and rounded it of by saying that my mum had the power to stop this from happening and she didnt. I think it was a kind of way to say that she was responsible mostly. To make us never forget this he tells each of us how badly we treated the girl it was really distressing. He then spoke out his final words which were very strong, powerful and deep and said there are many people still out there just like Eva Smith. He then walked out and slammed the door and that was it he had gone. I was still quietly crying thinking over and over again why did this happen? Then Eric and dad just started of worrying about the money stolen and it looked like they tried to pretend that nothing happened. No one learned anything from their mistakes.  I then started to think of the actual inspector; was he who we though he was? I didnt really think it mattered but I was just interested to find out. My dad said though that was really important to see if he was but I disagreed. I felt he was just not trying to face the facts. At the end of the day it doesnt matter if he was a police inspector or not the point is that he made us confess what we did wrong. Then we all started to talk about the police inspector; how strange he was. And then Gerald walked in I told him that we all got in trouble and then we all continued talking about how strange he was until Gerald comes up with something. We all waited for him to say it and stared at him. He said slowly that he wasnt a police inspector he sounded quite sure. There was even more evidence, he said he met a police sergeant and asked him if he knew an inspector Goole. Gerald described the chap to him and the sergeant said that he swore there was no one of that name or like him on the force. My dad then rang the chief constable to once more make sure, there was no inspector by the name of Goole. Everybody was a bit happier but I still thought in the end we all were responsible for a young girls death. I supported Eric at this time he said what I thought and what the rest dont understand that the girl is still dead and they are pretending that she isnt.  After a while of constant arguing Gerald comes up with something. He thought that the there was no real Eva Smith who changed her name to Daisy Renton and committed suicide. All he thought was that he was taking about different girls and that the photos were shown individually because of that reason. I was a bit less sad but I still could not forget the fact that all of us said what actually happened. Then Gerald phoned the infirmary to make sure there was no girl who committed suicide in the infirmary, as we waited patiently it was not true there was no dead girl there. I and Eric was still upset because we still did wrong things and we were aware of the consequences- at the end of the day we still harmed people and it was something that could have been real. I could still remember the inspector, his eyes how he glanced upon me, his voice, how he made me feel, his presence, all of it frightened me still. Everyone was happy again, smiling, triumphing and Gerald trying to propose to me again by saying its all over! But obviously I thought it was too soon and had to think about it. Everyone was laughing thinking that it was stupid and being amused until all the cheerfulness from everyone got interrupted by the ringing, the ringing of the phone. Dad picked the phone up; everyone was waiting anxiously due to all the events that took place. There was a deadly silence, I got scared. Dad put the phone down to say the fateful news. It was the police, a girl died in the infirmary after swallowing some disinfectant and a police inspector is on his way. That guilty conscience started to get bigger but the atmosphere was not only surrounded with that but also confusion and terror. My heart started to race up again, I was worried, I panicked and was confused. I thought of the situation again trying to make sense it just startled me even more. How did the so called inspector no about Eva!!?!

Zinc finger nuclease technology and its potential for modelling and treating disease The WritePass Journal

Zinc finger nuclease technology and its potential for modelling and treating disease Introduction Zinc finger nuclease technology and its potential for modelling and treating disease IntroductionMechanisms of DNA double strand break repair  Ã‚   Gene edition using ZFNs gene disruption and gene correction Gene addition Therapeutic applications of ZFNsLimitations of ZFNsReferences Related Introduction Methods to introduce site specific, stable modifications in complex genomes hold great potential, not only for the study of gene function but also for biotechnological and therapeutic applications (Sollu et al., 2010).   A promising new approach is based on zinc-finger nucleases (ZFNs), artificially constructed endonucleases that are designed to make a double strand break in a pre-determined genomic target sequence. This can then be followed by the generation of desired modifications during subsequent DNA repair. ZFNs are engineered to contain a DNA binding domain, composed of zinc finger proteins, and a non-specific endonuclease domain derived from the FokI restriction enzyme (Urnov et al., 2010). The zinc finger protein region provides a ZFN with the ability to bind to a discrete base sequence. Each zinc finger domain consists of   ÃŒ ´ 30 amino acids which fold into a ÃŽ ²ÃŽ ²ÃŽ ± structure, this is stabilised by chelation of a zinc ion by the conserved Cys2-His2 residues (Durai et al., 2005). Each domain recognises and binds to approximately 3bp of DNA. Binding to longer sequences is achieved by linking several of these zinc fingers in tandem to form zinc finger proteins. As the catalytic FokI domain must dimerise to induce a double strand break (Vanamee et al., 2001), two different ZFN subunits are designed that bind the sequence of interest in the opposite orientation and with the correct spacing. The combined target sequence is sufficient in length to be statistically unique, even in complex genomes (Sollu et al., 2010) (figure 1). ZFNs have been proven to work successfully in Arabidopsis thaliana (Zhang et al., 2010), Caenorhabditis elegans, Drosophila melanogaster (Carroll et al., 2008), zebrafish (Doyon et al., 2008), rats (Mashimo et al., 2010) and human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) (Zou et al., 2009). Mechanisms of DNA double strand break repair  Ã‚   All eukaryotic cells have effective mechanisms to repair double strand breaks in DNA. The two primary repair pathways are non-homologous end joining (NHEJ) and homologous directed repair (HDR) (Jackson and Bartek, 2009). These highly conserved pathways can be exploited to generate a defined genetic outcome across a wide range of cell types (Urnov et al., 2010). In NHEJ, the two broken ends are simply ligated back together. If the double strand break is complex, creating ends that are not compatible then repair by NHEJ will be mutagenic; the repaired DNA will contain small insertions or deletions at the site of the break, resulting in gene inactivation (Durai et al., 2005). If a double stranded oligonucleotide is provided with overhangs (sticky ends) complementary to those left by the ZFNs, it will be ligated into the chromosome, this approach can be used to add tags to endogenous genes. Alternately, two simultaneous double strand breaks made on the same chromosome can lead to a deletion of the entire intervening stretch (Lee et al., 2010) (figure 2). The other major repair pathway is HDR, a form of homologous recombination that faithfully copies the genetic information from a DNA molecule of related sequence. In HDR the 5Ê ¹ ends of the double strand break are resected to generate 3Ê ¹ single stranded tails, allowing strand invasion by donor DNA, which serves as a template for DNA replication (Durai et al., 2005). In normal double strand break repairs the DNA donor is the sister-chromatid, therefore the template is identical to the damaged DNA, resulting in a perfect form of repair. In gene targeting an exogenous donor DNA template is provided (usually an episomal or linear extrachromosomal donor) in combination to the ZFNs. If the donor DNA specifies solely a single nucleotide change, such as a restriction fragment length polymorphism (RFLP) encoding a novel allele, this will result in gene correction, that subtly edits the endogenous allele (Urnov et al., 2005). HDR can also be used for the addition of genes, if the donor pro vided carries an open reading frame (ORF), a transgene or even multiple trasngenes at the position corresponding to the site of the break, the sequence will be transferred to the chromosome (Moehle et al., 2007) (figure 2). Figure 2 | Types of genome editing made possible using ZFNs. The two primary repair pathways: non-homologous end joining (NHEJ) and homologous directed repair (HDR) with the different outcomes that can result from the introduction of a site specific DNA double strand break. Adapted from (Urnov et al., 2010). Gene edition using ZFNs gene disruption and gene correction The simplest means of gene editing is gene disruption, which takes advantage of errors introduced during DNA repair to disrupt or abolish the function of a gene or genomic region. Gene knockout (KO) is an affective tool for analysing gene function and generating model animals that recapitulate genetic disorders. Using ZFN technology, Mashimo et al., 2010 created knockout rats with X-linked Server Combined Immunodeficiency (X-SCID). They injected mRNAs encoding ZFNs designed to target the rat interleukin 2 receptor gamma (II2rg) locus, where orthologous human and mouse mutations cause X-SCID, into the pronucleus of fertilised rat oocytes. They found that the offspring carried a variety of deletion/insertion mutations, most of which were expressed as frameshift or splicing errors, resulting in no or very little expression of II2rg mRNA. The ZFN modified founders faithfully transmitted their genetic changes to the next generation along with the SCID phenotype (Mashimo et al., 2010). The X-SCID rats generated in studies such as this can be valuable in vivo tools for pre-clinical testing during drug development or gene therapy as well as model systems for examining the treatment of xenotransplanted malignancies. Another approach, gene correction allows the transfer of single nucleotide changes from a DNA donor to the chromosome following a ZFN induced double strand break. Urnov et al., 2005 designed ZFNs directed against the X-linked SCID mutation hotspot in the interleukin-2 receptor-ÃŽ ³ (IL2RÃŽ ³) gene. Using the ZFNs on K562 cell lines, they found that ~20% of the population carried a modification at the endogenous loci and about 7% of the cells were homozygous for the donor specified genotype, which was accurately reflected at the mRNA and protein levels. The modified cells were found to be stable for extended periods in cell culture while transcriptionally and translationally manifesting their new genotype (Urnov et al., 2005). Gene addition Transgenesis of human cells is used in functional genomics, proteomics and protein structure-function studies, and is routinely accomplished by random integration combined with drug selection. Expression of a randomly integrated transgene can be unpredictable and tends to be unstable over time due to epigenetic effects (DeKelver et al., 2010). The precisely placed double strand break induced by ZFNs can stimulate integration of long DNA stretches into a predetermined genomic location, resulting in site-specific gene addition. Moehle et al., 2007 introduced ZFNs directed against the interleukin-2 receptor-ÃŽ ³ (IL2RÃŽ ³) gene (exon 5), in combination with a DNA donor carrying a 12bp tag and a 900bp open reading frame (ORF), flanked by locus specific homology arms into HEK293 cells. After 72 hours, ~5% of the chromatids had acquired the transgene between the ZFN recognition sites (Moehle et al., 2007). ZFNs have also been used in human EPCs and iPSC to efficiently target a drug resistance marker to a specific gene. Hockemeyer et al., 2009, used ZFNs specific for the OCT4 (POU5F1) locus and a donor constructs containing a splice acceptor (SA) followed by an enhanced green fluorescent protein (eGFP)-2A-puromycin cassette. They reported expression of two proteins, a fusion protein comprising the first 132 amino acids of human OCT4 fused to eGFP (OCT4EX1-eGFP) and puromycin N-acetyltransferase, both under the control of the endogenous OCT4 promoter, therefore generating reporter cells which can monitor the pluripotent state of human ESCs (Hockemeyer et al., 2009). Therapeutic applications of ZFNs Site specific manipulation of the genome by ZFNs has revolutionised biology and holds great promise for molecular medicine (Lombardo et al., 2007). For example a corrected allele of a disease causing gene could be curative in several monogenetic diseases. Alternatively, the knockout of a gene encoding a virus receptor could be shown to eliminate rather than merely reduce infection. ZFN mediated gene disruption is the first ZFN based approach that has been taken to clinical trails, specifically for the treatment of glioblastoma (NCT01082926) and HIV (NCT00842634 and NCT01044654). In glioblastoma phase I clinical trials, the glucocorticoid receptor gene is disrupted by ZFNs as part of a T cell based cancer immunotherapy (Urnov et al., 2010). In the HIV trials, ZFNs targeting the chemokine (C-C motif) receptor type 5 (CCR5) gene have been delivered via adenoviral vector to isolated T cells from subjects. The CCR5 protein is required for certain common types of HIV infection to enter into and infect T cells. The ZFN mediated CCR5 knockout T cells then are returned to the subject.   (Perez et al., 2008). An advantage of using ZFN technology is that it creates a fully penetrant, heritable gene knockout that will persist for the lifetime of that cell and its progeny, therefore removing the need for persistent therapeutic exposure. Limitations of ZFNs A potential limitation of the ZFN targeting approach is off-target DNA breaks induced at related sequences elsewhere in the genome, which may cause unpredictable genotoxic. To overcome this, ZFNs can be designed to with longer DNA recognition sites such as 12bp-18bp, which upon dimerisation of the FokI nuclease domain will recognise a 24bp-23bp sequence (such sites are rare even in complex genomes). This alongside bioinformatic tools such as SELEX (systematic evolution of ligands by exponential enrichment) can be used determine the specificity for a ZFN DNA binding domain and generate a rank order of potential off-target site with highest similarity (Tuerk et al., 1990).   Another challenge when designing ZFNs is the choice delivery system (DNA, RNA or viral), the ideal method has proven to be dependent on cell type. Lombardo et al., 2007 found that integrase-defective lentiviral vectors (IDLV) support functional delivery of both ZFNs and donor DNA templates to a variety of cell ty pes, including haematopoietic progenitors and embryonic stem cells (Lombardo et al., 2007). Aside from the various limitations, ZFN technology has allowed site specific genome editing to become established in human cells and a number of model organisms, opening the door to a powerful range of new experimental and therapeutic possibilities. References Carroll, D., Beumer, K. J., Morton, J. J., Bozas, A. and Trautman, J. K. (2008) Gene targeting in Drosophila and Caenorhabditis elegans with zinc-finger nucleases, Methods Mol Biol, 435, pp. 63-77. DeKelver, R. C., Choi, V. M., Moehle, E. A., Paschon, D. E., Hockemeyer, D., Meijsing, S. H., Sancak, Y., Cui, X., Steine, E. J., Miller, J. C., Tam, P., Bartsevich, V. V., Meng, X., Rupniewski, I., Gopalan, S. M., Sun, H. C., Pitz, K. J., Rock, J. M., Zhang, L., Davis, G. 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