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.
Recombinant DNA technology: Sub cloning of cDNA molecule CIH-1 into plasmid vector pUC19, transformation of XLI-Blue Ecoli & restriction mapping.
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.
Shapiro, L. J. and Cederbaum, S. 1984. Recombinant DNA in Medicine. Western Journal of Medicine, 141 (2), p. 210. Available at: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1021739/ [Accessed: 10 Feb 2014].
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...
Gene expression is the ability of a gene to produce a biologically active protein. This process is regulated by the cells of an organism, it is very important to the survival of organisms at all levels. This is much more complex in eukaryotes than in prokaryotes. A major difference is the presence in eukaryotes of a nuclear membrane, which prevents the simultaneous transcription and translation that occurs in prokaryotes. Initiation of protein transcription is started by RNA polymerase. The activity of RNA polymerase is regulated by interaction with regulatory proteins; these proteins can act both positively, as activators, and negatively as repressors. An example of gene regulation in cells is the activity of the trp operon. The trp operon encodes the genes for the synthesis of tryptophan. This type of gene, like the lac operon, is regulated by a repressor that binds to the operator sequences. The activity of the trp repressor is enhanced when it binds tryptophan; in this capacity, tryptophan is known as a corepressor. Since the activity of the trp repressor is enhanced in the presence of tryptophan, the rate of expression of the trp operon is graded in response to the level of tryptophan in the cell. Another example of gene regulation in cells is gene amplification. This is a Technique by which selected DNA from a single cell can be duplicated indefinitely until there is a sufficient amount to analyse by conventional genetic techniques.
Li, Julang. "Mechanisms Involved in Targeted Gene Replacement in Mammalian Cells." Genetics. Vol. 156, 809-821. Oct. 2000.
During the summer of 2013 I began a project with my Physics Department in the field of nuclear science under the guidance of Dr. Mohammad Hannan. The project, an analysis of samples from a local canal via Neutron Activation Analysis (NAA), was a joint investigation between The University of Texas Pan American (UTPA) and The University of California Irvine (UCI). A
Rubanyi, G. (2001). The Future of Human Gene Therapy. Molecular Aspects of Medicine. 22,3, pp. 113-142.
Shi, Y., & Zou, M. (2008). Progress in gene therapy research. In J. L. Lewis (ED.), Gene therapy and cancer research progress (pp. 23-130). New York, NY: Nova Science Publishers, Inc.
Transcription occurs within the cytoplasm, and translation also occurs in the cytoplasm within the bacterial cell. However, within the eukaryotic cell the process of transcription occurs within the nucleus and translation occurs within the cytoplasm. The separation of the two processes is due to the fact that the nucleus is where DNA is contained in the eukaryotic cell, and the process of transcription changes the state of mRNA in order to escort the pre mRNA out of the nucleus and into the cytoplasm. During the initiation stage of transcription, the eukaryotic cells have a slightly more complex process. Eukaryotes require multiple transcription factors to bind to the DNA before the RNA polymerase II can attach. The attachment of the multiple transcription factors and the RNA polymerase II enzyme creates the transcription initiation complex, none of which occurs within the bacterial cell. After the transcription process, the eukaryotic cells requires some alteration to the mRNA strand in order to escort it out of the nucleus and into the cytoplasm. The addition of the 5’ cap and the 3’ cap is necessary in order to exit the nucleus; however, none the post-transcription modifications are needed for the bacterial cells. This is due to the previously stated reason of the eukaryotic cell having compartmentalization within the cell, while the bacterial cells have no nucleus holding the cell’s
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).
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
The Future of Gene Therapy. Biotechnol Healthc. 2005 Jun; 2(3): 52-54, 56-60. Retrieved from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3564347/
SCNT in terms of Therapeutic Cloning has revolutionized the way we now approach genes as the knowledge discovered about this process can now be used for both research and clinical applications including the use of SCNT product as a vector for gene delivery, the creation of animal models of human diseases, and cell replacement therapy in regenerative medicine. This technology has the potential to resolve human paralysis and has been proved to have the ability to regenerate spinal cord tissue as shown by Deshpande et al who transferred motor neurons derived from ESC to rats with a severed spinal cord. The process of SCNT, similar to that of reproductive cloning, includes the desired host oocyte to be obtained and immobilized through light suction exerted by a pipette tip. A glass needle is used to remove a small piece of the thick transparent membrane surrounding the ovum and is reinserted through this puncture to extract the polar body and the oocyte nuclei. The incorporation of the somatic nuclei into the enucleated oocyte can be done through electro fusion, which is the process of an electric pulse to integrate a mammalian cell into the oocyte. Alternatively, a somatic nucleus can be injected into the perivitelline space, which is the fluid-filled region between the zona pellucida and the cell membrane of an oocyte or fertilized
In a diabetic family with an IPF-1 frame shift mutation a protein has been shown to be generated as a result of translation re initiation. And this translation re initiation was shown to exert a dominant-negative effect on the activation of transcription by the wild-type protein. Despite