Gene knockout: A gene knockout (KO) is a genetic technique in which an organism is engineered to carry genes that have been made inoperative. However, KO can also refer to the gene that is knocked out or the organism that carries the gene knockout. Those organisms carrying such genes are known as knockout organisms or simple knockouts. In summary, KO is an organism in which a single gene of choice or interest is either inactivated or knockout in a manner that leaves all other genes unaffected and the best way to delineate the function of gene by homologous recombination method. The removed gene from the cell's DNA is impossible to be expressed. There will be absolutely none of the gene product in the cell.
Difference between gene knock in and knockout: Gene knock in is the opposite of gene knockout, hence the name. It involves replacing genes with new ones or even inserting the new ones without removing existing ones. This is also done with restriction enzymes. Gene knock in-introduce a new gene to your cell's DNA, which wasn't there before. This means there will now be a new gene product in your cell that wasn't there before. While, gene knockout is removing a gene from the cell's DNA so that it is impossible for it to be expressed. There will be absolutely none of the gene product in the cell.
Some knock in strategies rely on the use of gene vectors with flanking sequences, termed loxP, that on exposure to an enzyme called Cre recombinase undergo reciprocal recombination, leading to the deletion of the intervening DNA. For example, a gene-targeting vector is constructed to delete a specific exon of a gene in embryonic stem cells. Several kilobases of DNA on either side of the target gene are cloned around a drug-selection marker. After the cloned DNA is introduced into the stem cells, positive and negative drug selection occurs in culture. The left panel shows a targeting vector that was constructed with loxP sequences flanking the positive drug-selection gene. Cre recombinase can delete the DNA sequence between the loxP sites, thereby deleting a specific gene in the embryonic stem cells.
Fig 1. Gene knockout and knock in mice.
Knock in mice are generated by replacement of an endogenous exon with one harboring a mutation of interest. The gene-targeting strategy is similar to that used for knockout mice, except that a replacement exon is exchanged with the endogenous exon. Cre-loxP strategies can delete most traces of the targeting vector. Once the desired stem-cell clone is selected, it is injected into a blastocyst, which is implanted into the uterus of a foster mother. If the gene-targeted stem cells contribute to germ cells in the chimeric mice, subsequent offspring will harbor the gene-targeted mutation (germ-line transmission). Knock in of segments of the human immunoglobulin gene into mouse genome enables mice to produce therapeutically useful humanized antibodies.
1. JP Manis. Knock Out, Knock In, Knock Down-Genetically Manipulated Mice and the Nobel Prize. N Engl J Med. 2007 Dec 13;357(24):2426-9.