Gene Knock-outs

Molecular Genetics

Gene Knock-outs

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One way of connecting points on a molecular map with points on a genetic map is to target the sequence in the chromosome for mutation by a knock-out strategy.

Facts

A DNA fragment has been cloned and its position on the molecular map of a chromosome established. We do not know what, if any, phenotype is associated with mutations in this fragment. Thus the fragment is not located on the genetic map.
In vitro, the DNA fragment is altered by insertion of a fragment containing a selectable marker.
The interrupted "gene" is introduced into cells. In some cells, homologous recombination results in the replacement of the resident "gene" with the interrupted "gene".

Using special cells and techniques, cells with recombination events can be grown into whole organisms, usually heterozygous for the interrupted "gene". By breeding, homozygotes are produced.
The homozygous transgenic individuals are known as knock-outs. They are examined to determine what, if any, phenotype is associated with mutations in the targeted "gene".
If a knock-out phenotype is found, the locus responsible can be placed on the genetic map by standard crossing and segregation analysis.

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Interpretations

The knock-out strategy can create a visually observable phenotypic trait at a locus whose corresponding position on the molecular map is known.
The knock-out strategy could be used to test whether an isolated gene functions as thought.
The knock-out strategy could be used to discover what a null (complete lack of gene function) phenotype for a gene is.
The knock-out strategy will not work when multiple genes with redundant function exist.
Panels of knock-out mutants can be created by random insertion of transposable elements.

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Further information

The availability of organisms lacking function in a specific gene assists deciphering how the gene product works.
There are many considerations in designing knock-out organisms
Another way of connecting points on a molecular map with points on a genetic map is the antisense strategy.

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E-mail inquiries to U. Melcher------------Last Updated: 25 August, 2005

A DNA fragment has been cloned and its position on the molecular map of a chromosome established. We do not know what, if any, phenotype is associated with mutations in this fragment. Thus the fragment is not located on the genetic map. In vitro, the DNA fragment is altered by insertion of a fragment containing a selectable marker. The interrupted "gene" is introduced into cells. In some cells, homologous recombination results in the replacement of the resident "gene" with the interrupted "gene".
		Using special cells and techniques, cells with recombination events can be grown into whole organisms, usually heterozygous for the interrupted "gene". By breeding, homozygotes are produced. The homozygous transgenic individuals are known as knock-outs. They are examined to determine what, if any, phenotype is associated with mutations in the targeted "gene". If a knock-out phenotype is found, the locus responsible can be placed on the genetic map by standard crossing and segregation analysis.