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Construction of a long hairpin RNA for knockdown of endogenous gene in budding yeast

Andersson, Andreas LU (2012) KEMR13 20121
Department of Chemistry
Abstract
RNA interference (RNAi) has recently been discovered in the budding yeast Saccharomyces castellii. As in other organisms, artificial RNAi could be a useful genetic tool in budding yeast, but the method needs first to be established. S. castellii is also a useful model for telomerase studies, and a knockdown of the telomerase catalytic subunit Est2p could be a new way of unrevealing its functions and interactions in the cell. The aim of this work was to construct a long hairpin RNA directed against the EST2 mRNA. A region of 172 nucleotides was amplified by PCR using two different primer pairs with different restriction sites to obtain two fragments that could be inserted into a vector in opposite directions. One of these fragments was... (More)
RNA interference (RNAi) has recently been discovered in the budding yeast Saccharomyces castellii. As in other organisms, artificial RNAi could be a useful genetic tool in budding yeast, but the method needs first to be established. S. castellii is also a useful model for telomerase studies, and a knockdown of the telomerase catalytic subunit Est2p could be a new way of unrevealing its functions and interactions in the cell. The aim of this work was to construct a long hairpin RNA directed against the EST2 mRNA. A region of 172 nucleotides was amplified by PCR using two different primer pairs with different restriction sites to obtain two fragments that could be inserted into a vector in opposite directions. One of these fragments was successfully inserted. The other fragment was inserted into pUC18 to facilitate amplification for insertion into the final vector. RNA was isolated from S. castellii; the method used for RNA isolation proved to be appropriate for future measurements of mRNA levels after knockdown experiments. (Less)
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author
Andersson, Andreas LU
supervisor
organization
course
KEMR13 20121
year
type
H2 - Master's Degree (Two Years)
subject
keywords
Biokemi
language
English
id
2863069
date added to LUP
2012-08-14 14:37:12
date last changed
2012-08-14 14:37:12
@misc{2863069,
  abstract     = {RNA interference (RNAi) has recently been discovered in the budding yeast Saccharomyces castellii. As in other organisms, artificial RNAi could be a useful genetic tool in budding yeast, but the method needs first to be established. S. castellii is also a useful model for telomerase studies, and a knockdown of the telomerase catalytic subunit Est2p could be a new way of unrevealing its functions and interactions in the cell. The aim of this work was to construct a long hairpin RNA directed against the EST2 mRNA. A region of 172 nucleotides was amplified by PCR using two different primer pairs with different restriction sites to obtain two fragments that could be inserted into a vector in opposite directions. One of these fragments was successfully inserted. The other fragment was inserted into pUC18 to facilitate amplification for insertion into the final vector. RNA was isolated from S. castellii; the method used for RNA isolation proved to be appropriate for future measurements of mRNA levels after knockdown experiments.},
  author       = {Andersson, Andreas},
  keyword      = {Biokemi},
  language     = {eng},
  note         = {Student Paper},
  title        = {Construction of a long hairpin RNA for knockdown of endogenous gene in budding yeast},
  year         = {2012},
}