A knockout mouse has had a particular gene inactivated, so the gene's product is not made. By seeing what goes wrong, scientists can work out what the gene normally does, and can create models of human diseases.Method
1. A non-functional copy of the target gene is made, often with a marker gene inserted into it.
2. The faulty gene is introduced into mouse embryonic stem cells. In a few cells it replaces the normal gene by homologous recombination. Today CRISPR-Cas9 is often used instead.
3. Cells with the knockout are selected using the marker and injected into an early mouse blastocyst.
4. The blastocyst is implanted into a surrogate mother. The pups are chimeric, containing a mixture of normal and knockout cells.
5. Chimeric mice whose gametes carry the knockout are bred, giving heterozygous offspring.
6. Heterozygous mice are crossed, and a quarter of their offspring are homozygous for the knocked-out gene.
The scientists who developed this method won the 2007 Nobel Prize.Uses
• Mice with the CFTR gene knocked out are a model of cystic fibrosis, used to test new treatments.
• If a knockout mouse develops many tumours, this suggests the normal gene acts as a tumour suppressor gene, as with p53.Limitations
• Mice are not humans, and the same gene may act differently.
• Some knockouts are lethal to the embryo, so nothing can be learned about the gene's role later in life.
• Animal welfare: many mice are bred and some suffer.
1. A non-functional copy of the target gene is made, often with a marker gene inserted into it.
2. The faulty gene is introduced into mouse embryonic stem cells. In a few cells it replaces the normal gene by homologous recombination. Today CRISPR-Cas9 is often used instead.
3. Cells with the knockout are selected using the marker and injected into an early mouse blastocyst.
4. The blastocyst is implanted into a surrogate mother. The pups are chimeric, containing a mixture of normal and knockout cells.
5. Chimeric mice whose gametes carry the knockout are bred, giving heterozygous offspring.
6. Heterozygous mice are crossed, and a quarter of their offspring are homozygous for the knocked-out gene.
The scientists who developed this method won the 2007 Nobel Prize.Uses• Mice with the CFTR gene knocked out are a model of cystic fibrosis, used to test new treatments.
• If a knockout mouse develops many tumours, this suggests the normal gene acts as a tumour suppressor gene, as with p53.Limitations
• Mice are not humans, and the same gene may act differently.
• Some knockouts are lethal to the embryo, so nothing can be learned about the gene's role later in life.
• Animal welfare: many mice are bred and some suffer.