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Report and Opinion 2015;7(12) 69 Report and Opinion 2015;7(12) http://www.sciencepub.net/report Gene Knockout Research Literatures Ma Hongbao 1, Margaret Young 2, Yang Yan 1 1 Brookdale Hospital, Brooklyn, New York 11212, USA; 2 Cambridge, MA 02138, USA [email protected] Abstract: A gene knockout is a genetic technique in which one of an organism's genes is made inoperative. Also known as knockout organisms or simply knockouts, they are used in learning about a gene that has been sequenced, but which has an unknown or incompletely known function. Researchers draw inferences from the difference between the knockout organism and normal individuals. The term also refers to the process of creating such an organism, as in "knocking out" a gene. The technique is essentially the opposite of a gene knockin. Knocking out two genes simultaneously in an organism is known as a double knockout. Similarly the terms triple knockout and quadruple knockouts are used to describe three or four knocked out genes, respectively. This article introduces recent research reports as references in the related studies. [Ma H, Young M, Yang Y. Gene Knockout Research Literatures. Rep Opinion 2015;7(12):69-92]. (ISSN: 1553- 9873). http://www.sciencepub.net/report. 7. doi:10.7537/marsroj071215.07. Keywords: gene; knockout; cell; life; research; literature Introduction with a mean dominance coefficient ~0.2. Alleles with A gene knockout is a genetic technique in large homozygous effects are more likely to be more which one of an organism's genes is made inoperative. recessive than are alleles of weaker effect. Our Also known as knockout organisms or simply approach allows us to quantify, for the first time, the knockouts, they are used in learning about a gene that substantial variance and skew in the distribution of has been sequenced, but which has an unknown or dominance coefficients. This heterogeneity is so great incompletely known function. Researchers draw that many population genetic processes analyses based inferences from the difference between the knockout on the mean dominance coefficient alone will be in organism and normal individuals. The term also refers substantial error. These results are applied to the to the process of creating such an organism, as in debate about various mechanisms for the evolution of "knocking out" a gene. The technique is essentially the dominance, and we conclude that they are most opposite of a gene knockin. Knocking out two genes consistent with models that depend on indirect simultaneously in an organism is known as a double selection on homeostatic gene expression or on the knockout. Similarly the terms triple knockout and ability to perform well under periods of high demand quadruple knockouts are used to describe three or four for a protein. knocked out genes, respectively. The gene approach to the pathogenesis of male infertility may bring about Aragona, M. and M. T. Valente "Genetic some strategies for the diagnosis and manage of the transformation of the tomato pathogen Pyrenochaeta condition. Gene knockout technology is the lycopersici allowed gene knockout using a split- mainstream method currently used in the study of marker approach." Curr Genet. 2015 May;61(2):211- gene function. NANOG expression in prostate cancer 20. doi: 10.1007/s00294-014-0461-y. Epub 2014 Nov is highly correlated with cancer stem cell 21. characteristics and resistance to androgen deprivation. Pyrenochaeta lycopersici, as other soil- The following introduces recent reports as transmitted fungal pathogens, generally received little references in the related studies. attention compared to the pathogens affecting the aerial parts of the plants, although causing stunt and Agrawal, A. F. and M. C. Whitlock "Inferences about important fruit yield reduction of agronomic relevant the distribution of dominance drawn from yeast gene crops. The scope of this study was to develop a system knockout data." Genetics. 2011 Feb;187(2):553-66. allowing to investigate the functional role of P. doi: 10.1534/genetics.110.124560. Epub 2010 Nov 23. lycopersici genes putatively involved in the corky root Data from several thousand knockout rot of tomato. A genetic transformation system based mutations in yeast (Saccharomyces cerevisiae) were on a split-marker approach was developed and tested used to estimate the distribution of dominance to knock out a P. lycopersici gene encoding for a lytic coefficients. We propose a new unbiased likelihood polysaccharide monooxygenase (Plegl1) induced approach to measuring dominance coefficients. On during the disease development. The regions flanking average, deleterious mutations are partially recessive, Plegl1 gene were fused with the overlapping parts of 69 Report and Opinion 2015;7(12) http://www.sciencepub.net/report hygromycin marker gene, to favour homologous Cbs(+/-/)Asm(+/-) and Cbs(+/-/)Asm(-/-) as well as recombination. We were able to obtain four mutants their Cbs wild type littermates were used to study the not expressing the Plegl1 gene though, when tested on role of Asm(-/-) under a background of Cbs(+/-) with a susceptible tomato cultivar, Plegl1 mutants showed hHcys. HPLC analysis revealed that plasma Hcys unaltered virulence, compared with the wild-type level was significantly elevated in Cbs heterozygous strain. The strategy illustrated in the present work (Cbs(+/-)) mice with different copies of Asm gene demonstrated for the first time that homologous compared to Cbs(+/+) mice with different Asm gene recombination occurs in P. lycopersici. Moreover, a copies. Cbs(+/-/)Asm(+/+) mice had significantly transformation system mediated by Agrobacterium increased renal Asm activity, ceramide production and tumefaciens was established and stable genetic O(2.)(-) level compared to Cbs(+/+)/Asm(+/+), while transformants have been obtained. The transformation Cbs(+/-/)Asm(-/-) mice showed significantly reduced systems developed represent important tools for renal Asm activity, ceramide production and O(2.)(-) investigating both the role of genes putatively level due to increased plasma Hcys levels. Confocal involved in P. lycopersici interaction with host plant microscopy demonstrated that colocalization of and the function of other physiological traits which podocin with ceramide was much lower in Cbs(+/- emerged to be genetically expanded from the recent /)Asm(-/-) mice compared to Cbs(+/-/)Asm(+/+) mice, genome sequencing of this fungus. which was accompanied by a reduced glomerular damage index, albuminuria and proteinuria in Cbs(+/- Banerjee, T., D. K. Jaijyan, et al. "Apicoplast triose /)Asm(-/-) mice. Immunofluorescent analyses of the phosphate transporter (TPT) gene knockout is lethal podocin, nephrin and desmin expression also for Plasmodium." Mol Biochem Parasitol. 2012 illustrated less podocyte damages in the glomeruli Nov;186(1):44-50. doi: from Cbs(+/-/)Asm(-/-) mice compared to Cbs(+/- 10.1016/j.molbiopara.2012.09.008. Epub 2012 Oct 3. /)Asm(+/+) mice. In in vitro studies of podocytes, The C3, C5, C6 type sugar phosphate hHcys-enhanced O(2.)(-) production, desmin transporters bring sugars inside apicoplast, thus expression, and ceramide production as well as providing energy, reducing power and elements like decreases in VEGF level and podocin expression in carbon to apicoplast. Plasmodium berghei has two C3 podocytes were substantially attenuated by prior type sugar phosphate transporters in the membrane of treatment with amitriptyline, an Asm inhibitor. In apicoplast: triose phosphate transporter (TPT) and conclusion, Asm gene knockout or corresponding phosphoenolpyruvate transporter (PPT). Here we enzyme inhibition protects the podocytes and report that P. berghei TPT knockout parasites failed to glomeruli from hHcys-induced oxidative stress and survive. However, PPT knockout parasite behaved injury. similar to the wild type in the blood stages. The absence of PPT in other life stages, leads to defects in Boshra, H., J. Cao, et al. "Generation of Recombinant the development of parasite and was required at both Capripoxvirus Vectors for Vaccines and Gene mosquito as well as liver stages. This study also Knockout Function Studies." Methods Mol Biol. underlines the essentiality of triose transporters for 2016;1349:151-61. doi: 10.1007/978-1-4939-3008- apicoplast and its downstream pathways. 1_10. The ability to manipulate capripoxvirus Boini, K. M., M. Xia, et al. "Acid sphingomyelinase through gene knockouts and gene insertions has gene knockout ameliorates hyperhomocysteinemic become an increasingly valuable research tool in glomerular injury in mice lacking cystathionine-beta- elucidating the function of individual genes of synthase." PLoS One. 2012;7(9):e45020. Epub 2012 capripoxvirus, as well as in the development of Sep 14. capripoxvirus-based recombinant vaccines. The Acid sphingomyelinase (ASM) has been homologous recombination technique is used to implicated in the development of generate capripoxvirus knockout viruses (KO), and is hyperhomocysteinemia (hHcys)-induced glomerular based on the targeting a particular viral gene of oxidative stress and injury. However, it remains interest. This technique can also be used to insert a unknown whether genetically engineering of ASM gene of interest. A protocol for the generation of a gene produces beneficial or detrimental action on viral gene knockout is described. This technique hHcys-induced glomerular injury. The present study involves the use of a plasmid which encodes the generated and characterized the mice lacking flanking sequences of the regions where the cystathionine
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