A gene knockout is a genetically engineered organism that carries one or more genes in its chromosomes that has been made inoperative. So far such organisms have been engineered chiefly for research purposes. Also known as knockout organisms or simply knockouts, their most direct use is for learning about a gene that has been sequenced, but has an unknown or incompletely known function. Researchers draw inferences from how the knockout differs from individuals in which the gene of interest has not been made inoperative. Knockout is also the process of creating such an organism, as in "knocking out a gene." An iconic image of genetic engineering; this 1986 autoluminograph of a glowing transgenic tobacco plant bearing the luciferase gene of the firefly strikingly demonstrates the power and potential of genetic manipulation. ... In biology and ecology, an organism (in Greek organon = instrument) is a complex adaptive system of organs that influence each other in such a way that they function as a more or less stable whole and have properties of life. ... This stylistic schematic diagram shows a gene in relation to the double helix structure of DNA and to a chromosome (right). ... Figure 1: Chromosome. ... In genetics and biochemistry, sequencing means to determine the primary structure (or primary sequence) of an unbranched biopolymer. ...

Method

Knockout is accomplished through a combination of techniques, beginning in the test tube with a plasmid, a bacterial artificial chromosome or other DNA construct, and proceeding to cell culture. Individual cells are genetically transformed with the construct and--for knockouts in multi-cellular organisms--ultimately fused with a stem cell from a nascent embryo. Figure 1: Schematic drawing of a bacterium with plasmids enclosed. ... A bacterial artificial chromosome (BAC) is a DNA construct, based on a fertility plasmid, used for transforming and cloning in bacteria, usually E. coli. ... A DNA construct is an artificially constructed segment of nucleic acid that is going to be transplanted into a target tissue or cell. ... Epithelial cells in culture, stained for keratin (red) and DNA (green) Cell culture is the term applied when cells are grown in a synthetic environment. ... Cells in culture, stained for keratin (red) and DNA (green). ... Transformation is the genetic alteration of a cell resulting from the introduction, uptake and expression of foreign genetic material (DNA or RNA). ... Mouse embryonic stem cells. ... Embryos (and one tadpole) of the wrinkled frog (Rana rugosa). ...

The construct is engineered to recombine with the target gene, which is accomplished by incorporating sequences from the gene itself into the construct. Recombination then occurs in the region of that sequence within the gene, resulting in the insertion of a foreign sequence to disrupt the gene. With its sequence interrupted, the altered gene in most cases will be translated into a nonfunctional protein, if it is translated at all. Recombination usually denotes a genetic event that occurs during the formation of sperm and egg cells (especially in areas of study of biology topics). ... part of a DNA sequence A DNA sequence (sometimes genetic sequence) is a succession of letters representing the primary structure of a real or hypothetical DNA molecule or strand, The possible letters are A, C, G, and T, representing the four nucleotide subunits of a DNA strand (adenine, cytosine, guanine... Translation is an activity comprising the interpretation of the meaning of a text in one language — the source text — and the production, in another language, of a new, equivalent text — the target text, or translation. ... A representation of the 3D structure of myoglobin, showing coloured alpha helices. ...

A conditional knockout allows gene deletion in a tissue specific manner.

Because recombination is a rare event in the case of most cells and most constructs, the foreign sequence chosen for insertion usually is a reporter. This enables easy selection of cells or individuals in which knockout was successful. In molecular biology, a reporter gene (often simply reporter) is a gene that researchers attach to another they wish to study in cell culture, animals or plants. ...

In diploid organisms, which contain two alleles for most genes, and may as well contain several related genes that collaborate in the same role, additional rounds of transformation and selection are performed until every targeted gene is knocked out. Diploid (meaning double in Greek) cells have two copies (homologs) of each chromosome (both sex- and non-sex determining chromosomes), usually one from the mother and one from the father. ... An allele is any one of a number of viable DNA codings of the same gene (sometimes the term refers to a non-gene sequence) occupying a given locus (position) on a chromosome. ...

Compare: knock-in; knockdown A gene knockdown, is a genetically modified organism that carries one or more genes in its chromosomes that has been made less active or had its expression reduced. ...

See also

• knockout mouse
• knockdown mouse
• germline
• gene silencing