Student Wiki on methodology

This Wiki is intended to collectively make the point on methodologies employed in research papers we analyze during the course. "Writers" are students who wish to contribute to a specific subject. Before contributing, please add your name in the "Writers group choice". When initiating a contribution, please indicate your name in brackets.


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Knocking-down and knocking-out genes

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Modified: 17 April 2019, 10:58 AM   User: Ilaria Ghia  → 

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Tentative subjects:

1. Transient siRNA/LNA-mediated knock-down

2. Stable shRNA knock-down

3. K.O. in cultured cells, alternatives

(Ilaria Ghia)

A knockout cell line (constitutive KO) is a model in which a target gene is completely inactivated and thus is not able to produce the protein it encodes for. 

The applications of this technology are many, and can be distinguished on the type of research it is used for:

  • Academic research: study of the main functions of a gene and/or protein, LOF (Loss-of-function) studies, in vitro recapitulation of human diseases;
  • Bio-pharmaceutical research: in vitro target validation, drug study and screening through mimicry of human diseases,study of safety, off-target activity and specificity of a drug.

As every technique, knockout cell lines show some strength and limitations. Three main advantages are:

  • Total absence of protein, excluding all the possible isoforms;
  • Since each cell line is matched with an isogenic (=genetically identical) control cell line, it is a reliable methodology;
  • Feasible in every genetic background or cell type.

By contrast, three weaknesses are:

  • Genetic compensation: the function of the KO protein can be supplied by the action of other related proteins;
  • Non-specific phenotype: the editing of a gene can sometimes affect the neighboring genes too;
  • Not very suitable for highly polyploid cell lines.