The major conclusion of this study is that our novel split-Cre complementation system introduces both temporal and special control of site specific recombination using Cre recombinase enzyme. This system solved many drawbacks have emerged during the extensive use of Cre recombinase in molecular biology. The complemented protein is almost as efficient as the Full CRE in the recombination activity (~95%). Moreover, each fragment lacks the recombinase activity. This system allows precise genetic manipulation. It has a special importance in neuroscience lacking selective promoter region for conditional knocking our specific gene at specific brain tissue. Many websites, such as GenePaint; a digital atlas for gene expression pattern in mouse embryo (http://www.genepaint.org/Frameset.html) (35), METscout (http://www.metscout.mpg.de/) (36), and GENESAT Brain Atlas of Gene Expression (http://www.gensat.org/cre.jsp) have been emerged. We can determine the suitable promoter that selectively expressed in the required tissue with the desired expression intensity among these promoters. Recently, the possibility of Cre complementation has been explored by several laboratories. It has advantages over temporal control of Cre recombinase activity. For example, preceding genetic marking of cells expressing the first fragment of Cre (nCre or cCre), by co-expressing EGFP. Afterwards, introduction of the second Cre fragment, to obtain the recombinase activity, will give a chance for direct comparison between the cellular morphology and dynamic behavior before and after genetic modification (37). Previous trials splitted CRE into two fragments such as (19-59, 60-343) (19) and (1-190, 191-343) (20) could restore maximum 35% of the full CRE recombinase a...
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... L. Spatial and temporal expression of the Cre gene under the control of the MMTV-LTR in different lines of transgenic mice. Transgenic Res. 2001; 10(6): 545-553.
44- O'Neill MT, Phuong T, Healer J, Richard D, Cowman AF. Gene deletion from Plasmodium falciparum using FLP and Cre recombinases: implications for applied site-specific recombination. Int J Parasitol. 2011; 41(1): 117-123.
45- Koblizek TI, Siehoff A, Pitt A. Systematic analysis of complex signal transduction pathways using protein fragment complementation assays. Methods Mol Biol. 2013; 986: 179-185.
46- Kozak M. Rethinking some mechanisms invoked to explain translational regulation in eukaryotes. Gene. 2006; 382: 1-11.
47- Smith AE, Kalderon D, Roberts BL, Colledge WH, Edge M, Gillett P, Markham A, Paucha E, Richardson WD. The nuclear location signal. Proc R Soc Lond B Biol Sci. 1985; 226(1242): 43-58.
A previous study, looked at by the researchers, stated that nuclear localization signals are what allow the RNA to enter the nucleus (Wu W, Pante N. 2009). This persuaded them to ask the question of whether or not there was a nuclear localization signal within a viral protein of HCRSV. The localization of P23 was then tested using a transient expression method. The results of their experiment showed that there was a strong signal detected in the nucleus of the Kenaf leaf samples. This proved that P23 was in fact localized in the nucleus and that a nuclear localization signal is present in P23 (Gao R, Liu P, Wong SM. 2012). It was also found that P23 has the ability to bind to carrier proteins that come into the nucleus. This showed that even if P23 was not localized in the nucleus, it could still enter. The mode of entry into the nucleus was discovered to by α-importin (Gao R, Liu P, Wong SM. 2012) . This was discovered by experimenting with a probe of anti-importin α antibody. α-importin was only detected in the protein extract of P23 in the nucleus of the HCRSV-infected Kenaf sample (Gao R, Liu P, Wong SM. 2012). Researchers concluded from their results of the experiments that α-importin, P23, and HCRSV RNA form a complex that enters the nucleus to begin replication of the
Recombinant DNA technology: Sub cloning of cDNA molecule CIH-1 into plasmid vector pUC19, transformation of XLI-Blue Ecoli & restriction mapping.
Ligating the EGFP cDNA into a pET41a (+) plasmid in order to create recombinant expression plasmids and run these ligations through gel electrophoresis to visualize the DNA and check the success of the ligations. Five ligation reactions were generated, two actual ligations and three controls, with a total final volume of 20uL each. NcoI and NotI are restriction endonucleases whose purpose are to reduce non-recombinant plasmids from forming and to prevent undesired rearrangements during ligation.
Takahashi, Y., et al. “Analysis of Promoter binding by the E2F and pRB Families In Vivo: Distinct E2F Proteins Mediate Activation and Repression.” Genes 14 (2000): 804-816.
3) And literature search of evidence for the functional properties of the genes of their homologues. The analyses will help to build an integrated picture which shows the genes in the region together with their expression profiles and functional properties. Such information can be used to guide our experimental strategy towards the identification of the interested gene and will provide a case-study for using bioinformatics approach to aid traditional genetic approach in gene identification.
A recombinant plasmid are created by first using an enzyme that can identify and isolate specifically which gene that need to be cut. They are call restriction enzymes or restriction endonucleases, and more than 100 of these enzymes have been isolated. After the human gene (gene of interest) that codes for the desire trait is located on the chromosome restriction enzyme does it job, by cutting out the gene from the DNA. Now, the two ends of the human gene will be those that will link up with the open ends of the plasmid. An enzyme, DNA ligase, is used to couple each end of the gene to the open ends of the plasmid; this thus restores the circular DNA molecule with the human gene. Now the plasmid, with the human gene, is reinserted into the bacteria. They are then cultured and produced in large quantities of identical bacteria carrying the human gene. Now, these bacteria produce the human protein coded for by the spliced human gene. The protein is then isolated and purified and are ready to be injected into patients (crop, etc.) (Gish 1998).
Plasmodium Falciparum is the causative parasite of malignant malaria, it is the most deadly strain of the malaria viruses. P. Falciparum is a eukaryotic protozoan parasite that is spread through vector transmission using mosquitoes. The Anopheles mosquito family accounts for the majority of transmission because of their tendency to target humans (WHO, 2014). Malaria accounts for approximately five hundred thousand deaths each year in environments such as sub-Saharan Africa and other temperate areas where the life cycle of the mosquito is longer allowing the parasite to develop properly (WHO, 2014). Malaria usually infects children in these areas but also commonly spreads to travellers to these areas.
Gene therapy works by introducing new and functioning genetic material to damaged genes to help it function and to produce beneficial proteins. If a gene is inserted directly into a cell, it usually will not function. So to complete this task, a vector, a modified virus is used to carry and deliver the new gene. There are two different categories of vectors than can be utilized in this process; recomb...
Society of Nuclear Medicine and Molecular Imaging (1954-2014). About Nuclear Medicine & Molecular Imaging. Retrieved from: http://www.snmmi.org/AboutSNMMI/Content.aspx?ItemNumber=6433
Li, Julang. "Mechanisms Involved in Targeted Gene Replacement in Mammalian Cells." Genetics. Vol. 156, 809-821. Oct. 2000.
The Gal4/UAS system is a common, powerful method used for studying gene expression, especially in model organisms (Lynd, 2012). This method can be used to directly study the phenotypes generated through transgene mis- or over-expression. It consists of two parts; the Gal4 gene that encodes the yeast transcription activator protein Gal4, and the UAS (upstream activation sequence) that is an enhancer to which Gal4 binds in order to activate gene transcription. This system was first found in the yeast Saccbaromyces cerevistae and has since been added into model organisms for the purpose of studying gene regulation (Laughon 1984). The Gal4 gene encodes for a protein that has been shown to be a regulator for Gal10 and Gal1 genes by binding to four loci located between the genes (Duffy 2002). The loci that the Gal4 binds to are defined as UASs, which are similar to the enhancers in eukaryotic organisms. Without the Gal4 binding, there would be no expression of the genes defined by the UAS (Giniger 1985).
Wen, Chuck K., Pamela L. Hudak, and Stephen W. Hwang. "Abstract." National Center for Biotechnology Information. U.S. National Library of Medicine, 06 Apr. 2007. Web. 13 Apr. 2014.
The process of translation is a major part of protein synthesis. There are many different components related the process of protein synthesis which include the large ribosomal unit, 60S and the small ribosomal unit,40S. As well as these are the messenger RNA, “mRNA coding”, transfer RNA , tRNA for amino acids and finally greater than 12 of the catalytic proteins which have be found to be eIFs (eukaryotic initiation factors). (Norton and Layman, 2006)These initiation factors are quite important in relation to Protein Synthesis and translation initiation. ...“These initiation factors guide the assembly of the ribosome on the mRNA and are responsive to short-term changes in the availability of energy, amino acids, and growth factors. Initiation factors provide the cell with sensitivity to environmental factors, including changes in diet, such as leucine availability, and physical activity.”... As well as this they also enable the cell to become sensitive to factors like the availability of
For instance, the muscle degenerative disease sarcopenia or muscular dystrophy could be benefited from the inhabitation of this gene (Lee 2004). In the two studies conducted in regards to this topic when this gene was suppressed there was also evidence of a decrease in fibrosis, which leads to promising hopes for regeneration of some degenerative diseases (Lee 2004). However, there are still many doubts and uncertainties surrounding these experiments (Lee 2004).
Campbell N. A., Reece L. A., Cain M. L., Wasserman S. A., Minorsky P. V. and Jackson R. B. (2008). Regulation of Gene Expression