User:Lmohamm9/sandbox

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Ectopic expression[edit]

Ectopic expression is the expression of a gene in a tissue where it is not normally expressed.[1] Ectopic expression also refers to the expression of a gene at a point in the cell cycle or in an organism's development when it is not normally expressed.[2] This can be caused by a disease, or it can be artificially produced as a way to help determine the function of that gene.[3] Gene up-regulation may mediate the development of structures in abnormal locations, resulting in ectopic expression.[4]

Ectopic expression systems[edit]

Ectopic expression of a gene can be done by introducing a transgene with a modified promoter into the target organism (transient or stable transfection) or by using the Gal4/UAS system.[5] As a result, use of ectopic expression systems can introduce a new phenotype which may be lethal.[6][7] If an extreme phenotype is to be expected, a promoter which is inducible should be used.[8][9][10]

Traditionally, ectopic expression of a gene using Drosophila melanogaster involves mutant generation via dominant mutations. This allows for the over expression and/or misexpression of the final functional gene product. Elaborate studies by the D. melanogaster research community gave rise to multiple ectopic expression systems available for use.[11]

With all D. melanogaster ectopic expression mechanisms, a promoter designed for the gene of interest is utilized to initiate transcription. The promoter results in either constitutive or regulated gene expression. In order to observe the effects between wild type and ectopically expressed genes, a transgenic line must be produced.[11]

D. melanogaster polyubiquitin and EF-1α promoters[edit]

Polyubiquitin and EF-1α promoters from D. melanogaster, have been utilized for uniform ectopic expression.[12] [13] EF-1α promoters allow for elevated levels of transcription while maintaining uniform expression.[11] On the other hand, use of polyubiquitin promoters such as the polyubiquitin-lacZ can be limited. The polyubiquitin-lacz promoter allows for uniform expression in early embryos and imaginal discs, not other developmental stages.[14]

Pwum2, a P-element polyubiquitin vector, has been proven effective in D. melanogaster ectopic expression. Pwum2 includes a polyubiquitin promoter combined with a myc tag. The vector itself includes two restriction enzyme sites (KpnI and Not1). The tag has a significant role in ectopic expression systems, allowing for the detection of encoded proteins in transformed organisms.[11]

Heat shock ectopic expression[edit]

Heat shock promoters, promoters from heat shock proteins (HSP),[15] have been used in D. melanogaster to regulate gene expression.[16][17][18] Compared to higher organisms, D. melanogaster temporal temperatures can be regulated, this allows manipulation of ectopic expression levels. In addition, altered durations of heat treatments can be used to modify levels of ectopic expression.[11]

There are three disadvantages when it comes to the heat shock ectopic expression system. Firstly, the transcription levels for some heat shock promoters is low under non-heat shock conditions. This is detrimental to the cell if the gene encodes a toxic product. Secondly, it is difficult to study tissue-specific functions when heat shock promoters are expressed in all cell types during an experiment.[11] Lastly, under heat shock conditions the heat shock promoter is active, however, endogenous cellular gene expression is disrupted. Decreased expression of specific regulatory genes due to heat treatment may result in mutant D. melanogaster phenotypes.[19]

Ectopic expression in the epigenome[edit]

Agouti gene[edit]

Varying fur coat colours in mice due to altered levels of ectopic expression involving the agouti gene.

The agouti gene in mice is responsible for the pigmentation of mouse coat patterns and hair follicle development. Ectopic expression of this gene is variable in mice and has been associated with the predisposition of tumours and obesity.[20] The mechanism of ectopic expression involves the epigenetic regulation of IAP (Intracisternal A-Particle), an insertion element located upstream of the agouti gene. When the IAP is demethylated in mice, ectopic expression is driven from a cryptic promoter encoded by the IAP insertion.[21] Minimal ectopic expression of the agouti gene results in mice with a brown fur coat, maximum ectopic expression of the agouti gene results in mice with a yellow fur coat.[22]

References[edit]

  1. ^ Schepers, Ute (2006). RNA Interference in Practice: Principles, Basics, and Methods for Gene Silencing in C. elegans, Drosophila, and Mammals. John Wiley & Sons. ISBN 3527604375.
  2. ^ Prelich, Gregory (2012-03-01). "Gene Overexpression: Uses, Mechanisms, and Interpretation". Genetics. 190 (3): 841–854. doi:10.1534/genetics.111.136911. ISSN 0016-6731. PMC 3296252. PMID 22419077.
  3. ^ Perdew, Gary H; Vanden Heuvel, John P; Peters, Jeffrey M, eds. (2007). Regulation of Gene Expression. Humana Press. p. 240. doi:10.1007/978-1-59745-228-1.
  4. ^ Sarnat, Harvey B; Curatolo, Paolo (2007). Malformations of the Nervous System: Handbook of Clinical Neurology. Newnes. p. 110. ISBN 0080559840.
  5. ^ Tuan, Rocky S.; Lo, Cecilia W. Developmental Biology Protocols. doi:10.1385/1592590667.
  6. ^ Moench, Thomas R.; Gendelman1, Howard E.; Clements, Janice E.; Narayan, Opendra; Griffin, Diane E. (1985-06-01). "Efficiency of in situ hybridization as a function of probe size and fixation technique". Journal of Virological Methods. 11 (2): 119–130. doi:10.1016/0166-0934(85)90035-7.{{cite journal}}: CS1 maint: numeric names: authors list (link)
  7. ^ Smith, Laura M.; Handley, Jane; Li, Yi; Martin, Helen; Donovan, Linda; Bowles, Dianna J. (1992-10-01). "Temporal and spatial regulation of a novel gene in barley embryos". Plant Molecular Biology. 20 (2): 255–266. doi:10.1007/BF00014493. ISSN 0167-4412.
  8. ^ Casey, James; Davidson, Norman (1977-06-01). "Rates of formation and thermal stabilities of RNA:DNA and DNA:DNA duplexes at high concentrations of formamide". Nucleic Acids Research. 4 (5): 1539–1552. doi:10.1093/nar/4.5.1539. ISSN 0305-1048. PMID 19730.
  9. ^ Hemmati-Brivanlou, A.; Frank, D.; Bolce, M. E.; Brown, B. D.; Sive, H. L.; Harland, R. M. (1990-10-01). "Localization of specific mRNAs in Xenopus embryos by whole-mount in situ hybridization". Development (Cambridge, England). 110 (2): 325–330. ISSN 0950-1991. PMID 1723941.
  10. ^ Martin, R.; Hoover, C.; Grimme, S.; Grogan, C.; Höltke, J.; Kessler, C. (1990-12-01). "A highly sensitive, nonradioactive DNA labeling and detection system". BioTechniques. 9 (6): 762–768. ISSN 0736-6205. PMID 2148679.
  11. ^ a b c d e f D'Avino, Pier Paolo; Thummel, Carl S. Ectopic expression systems in Drosophila. pp. 129–142. doi:10.1016/s0076-6879(99)06009-7.
  12. ^ Lee, H. S.; Simon, J. A.; Lis, J. T. (1988-11-01). "Structure and expression of ubiquitin genes of Drosophila melanogaster". Molecular and Cellular Biology. 8 (11): 4727–4735. doi:10.1128/MCB.8.11.4727. ISSN 0270-7306. PMID 2463465.
  13. ^ Ackermann, Ruedi; Brack, Christine (1996-06-01). "A Strong Ubiquitous Promoter-Enhancer for Development and Aging of Drosophila melanogaster". Nucleic Acids Research. 24 (12): 2452–2453. doi:10.1093/nar/24.12.2452. ISSN 0305-1048. PMID 8710521.
  14. ^ Vincent, J. P.; Girdham, C. (1997-01-01). "Promoters to express cloned genes uniformly in Drosophila". Methods in Molecular Biology (Clifton, N.J.). 62: 385–392. doi:10.1385/0-89603-480-1:385. ISSN 1064-3745. PMID 9108535.
  15. ^ Calderwood, Stuart K.; Sherman, Michael Y.; Ciocca, Daniel R., eds. (2007). Heat Shock Proteins in Cancer. Springer Netherlands. p. 58. doi:10.1007/978-1-4020-6401-2.
  16. ^ Struhl, Gary (1985-12-19). "Near-reciprocal phenotypes caused by inactivation or indiscriminate expression of the Drosophila segmentation gene ftz". Nature. 318 (6047): 677–680. doi:10.1038/318677a0.
  17. ^ Lis, J. T.; Simon, J. A.; Sutton, C. A. (1983-12-01). "New heat shock puffs and beta-galactosidase activity resulting from transformation of Drosophila with an hsp70-lacZ hybrid gene". Cell. 35 (2 Pt 1): 403–410. ISSN 0092-8674. PMID 6418386.
  18. ^ Schneuwly, S.; Klemenz, R.; Gehring, W. J. (1987-03-26). "Redesigning the body plan of Drosophila by ectopic expression of the homoeotic gene Antennapedia". Nature. 325 (6107): 816–818. doi:10.1038/325816a0. ISSN 0028-0836. PMID 3821869.
  19. ^ Mitchell, HK; Peterson, NS (1984). Gerald A, Kerkut; Gilbert, Lawrence Irwin (eds.). Comprehensive Insect Physiology, Biochemistry and Pharmacology. Oxford: Pergamon Press. p. 347. ISBN 0080268501.
  20. ^ Michaud, E. J.; van Vugt, M. J.; Bultman, S. J.; Sweet, H. O.; Davisson, M. T.; Woychik, R. P. (1994-06-15). "Differential expression of a new dominant agouti allele (Aiapy) is correlated with methylation state and is influenced by parental lineage". Genes & Development. 8 (12): 1463–1472. ISSN 0890-9369. PMID 7926745.
  21. ^ Whitelaw, Emma; Morgan, Hugh D.; Sutherland, Heidi G.E.; Martin, David I.K. "Epigenetic inheritance at the agouti locus in the mouse". Nature Genetics. 23 (3): 314–318. doi:10.1038/15490.
  22. ^ Niculescu, Mihai D.; Haggarty, Paul (2011-03-25). Nutrition in Epigenetics. John Wiley & Sons. ISBN 9780470959800.
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