Gene knockout

A gene knockout (abbreviation: KO) is a genetic technique in which one of an organism's genes is made inoperative ("knocked out" of the organism). Also known as knockout organisms or simply knockouts, they are used in learning about a gene that has been sequenced, but which has an unknown or incompletely known function. Researchers draw inferences from the difference between the knockout organism and normal individuals. The term also refers to the process of creating such an organism, as in "knocking out" a gene. The technique is essentially the opposite of a gene knockin. Knocking out two genes simultaneously in an organism is known as a double knockout (DKO). Similarly the terms triple knockout (TKO) and quadruple knockouts (QKO) are used to describe three or four knocked out genes, respectively. Method

A laboratory mouse in which a gene affecting hair growth has been knocked out (left), is shown next to a normal lab mouse. 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 transfected with the DNA construct. Often the goal is to create a transgenic animal that has the altered gene. If so, embryonic stem cells are genetically transformed and inserted into early embryos. Resulting animals with the genetic change in their germline cells can then often pass the gene knockout to future generations. To create knockout moss, transfection of protoplasts is the preferred method. Such transformed Physcomitrella-protoplasts directly regenerate into fertile moss plants. Already eight weeks after transfection the plants can be screened for gene targeting via PCR.

Wild-type Physcomitrella and knockout-mosses: Deviating phenotypes induced in genedisruption library transformants. Physcomitrella wild-type and transformed plants were grown on minimal Knop medium to induce differentiation and development of gametophores. For each plant, an overview (upper row, scale bar corresponds to 1 mm) and a close-up (bottom row, scale bar equals 0.5 mm) is shown. A, Haploid wild-type moss plant completely covered with leafy gametophores and close-up of wild-type leaf. B-D, Different Mutants. 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.

A knockout mouse (left) that is a model of obesity, compared with a normal mouse. A conditional knockout allows gene deletion in a tissue or time specific manner. This is done by introducing short sequences called loxP sites around the gene. These sequences will be introduced into the germ-line via the same mechanism as a knock-out. This germ-line can then be crossed to another germline containing Cre-recombinase which is a viral enzyme that can recognize these sequences, recombines them and deletes the gene flanked by these sites. Because the desired type of DNA recombination is a rare event in the case of most cells and most constructs, the foreign sequence chosen for insertion usually includes a reporter. This enables easy selection of cells or individuals in which knockout was successful. Sometimes the DNA construct inserts into a chromosome without the desired homologous recombination with the target gene. To eliminate such cells, the DNA construct often contains a second region of DNA that allows such cells to be identified and discarded. 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. Selective breeding may be required to produce homozygous knockout animals. Knock-in is similar to knock-out, but instead it replaces a gene with another instead of deleting it.

Use Knockouts are primarily used to understand the role of a specific gene or DNA region by comparing the knockout organism to a wildtype with a similar genetic background. Knockouts organisms are also used as screening tools in the development of drugs, to target specific biological processes or deficiencies by using a specific knockout, or to understand the mechanism of action of a drug by using a library of knockout organisms spanning the entire genome, such as in Saccharomyces cerevisiae. Gene knockdown refers to techniques by which the expression of one or more of an organism's genes is reduced, either through genetic modification (a change in the DNA of one of the organism's chromosomes) or by treatment with a reagent such as a short DNA or RNA oligonucleotide with a sequence complementary to either an mRNA transcript or a gene. If genetic modification of DNA is done, the result is a "knockdown organism". If the change in gene expression is caused by an oligonucleotide binding to an mRNA or temporarily binding to a gene, this results in a temporary change in gene expression without modification of the chromosomal DNA and is referred to as a "transient knockdown". In a transient knockdown, the binding of this oligonucleotide to the active gene or its transcripts causes decreased expression through blocking of transcription (in the case of gene-binding), degradation of the mRNA transcript (e.g. by small interfering RNA (siRNA) or RNase-H dependent antisense) or blocking either mRNA translation, pre-mRNA splicing sites or nuclease cleavage sites used for maturation of other functional RNAs such as miRNA (e.g. by Morpholino oligos or other RNase-H independent antisense). The most direct use of transient knockdowns is for learning about a gene that has been sequenced, but has an unknown or incompletely known function, an experimental approach known as reverse genetics. Researchers draw inferences from how the knockdown differs from individuals in which the gene of interest is operational. Transient knockdowns are often used in developmental biology because oligos can be injected into single-celled zygotes and will be present in the daughter cells of the injected cell through embryonic development.[3] So far knockdown organisms with permanent alterations in their DNA have been engineered chiefly for research purposes. Also known simply as knockdowns, these organisms are most commonly used for reverse genetics, especially in species such as mice or rats for which transient knockdown technologies cannot easily be applied.

In molecular cloning and biology, a Knock-in (or Gene knock-in) refers to a genetic engineering method that involves the insertion of a protein coding cDNA sequence at a particular locus in an organism's chromosome.[1] Typically, this is done in mice since the technology for this process is more refined, and because mouse embryonic stem cells are easily manipulated. The difference between knock-in technology and transgenic technology is that a knock-in involves a gene inserted into a specific locus, and is a "targeted" insertion.

A common use of knock-in technology is for the creation of disease models. It is a technique by which scientific investigators may study the function of the regulatory machinery (e.g. promoters) that governs the expression of the natural gene being replaced. This is accomplished by observing the new phenotype of the organism in question.The BACs and YACs are used in this case so that large fragments can be transferred. Conditional gene knockout is a relatively new technique, an offshoot of gene knockout technology with the exception that a specific target gene is eliminated from a single organ in the body rather than the whole body as gene knockout technology would entail. Thus this allows for more sophisticated experiments than traditional gene knockout studies and hence more useful and practical in its approach. There are several advantages conferred by this technique over the conventional type in that such genetically modified mice are not only able to survive longer but also, the technique overall is more clean and scientifically precise. The most commonly used technique for conditional gene knockout is the Cre-Lox recombination system. Cre recombinase is known to be expressed in only a few target cell types and hence the researcher would be typically able to decide where exactly the gene in question is to be knocked out thus allowing for a greater degree of freedom and scientific accuracy. Latest advances A team of researchers from UT Southwestern Medical Center have successfully knocked out a specific gene in mouse brain thought to be involved in the onset of Alzheimer's disease which codes for the enzyme cyclin-dependent kinase 5 (Cdk5). Such genetically-engineered mice were found to be 'smarter' than normal mice and were able to handle complex tasks more intelligently compared to 'normal' mice bred in the laboratory.

Homologous Recombination Method (and Knockout Mouse)

When an investigator wants to replace one allele with an engineered construct but not affect any other locus in the genome, then the method of choice is homologous recombination. To perform homolgous recombination, you must know the DNA sequence of the gene you want to replace (figure 1). With this information, it is possible to replace any gene with a DNA construct of your choosing. The method has a few more details than will be illustrated on this page, but the essential information is retained.

Figure 1. Diagram of gene targeted for replacement by an engineered construct. The coding sequence is illustrated by the box with flanking upstream and downstream DNA sequences provided. The arrows pointing away from the targeted gene represent the continuous chromosomal DNA.

The next step is to design and fabricate the DNA construct you want to insert into the chromosome in place of the wild-type allele. This construct may contain any DNA sequence of your choosing which means you can insert different alleles (both functional and non-functional ones), different genes or reporter genes (e.g. antibiotic resistance or green fluorescent protein). Regardless of what you want to insert, you must include some flanking DNA that is identical in sequence to the targeted locus (figure 2). In addition to the positive selection marker (e.g. antibiotic resistance) often a negative selection marker (e.g. thymidine kinase, tk) is added to the replacement vector. The negative marker is outside the region of sequence similarity between the vector and the targeted locus.

Fiugre 2. Diagram of engineered construct that will be used to replace the wild-type allele. The upstream and downstream flanking DNA sequences are identical to those which flank the targeted locus. The negative marker tk is shown in to the right of the region of sequence similarity.

The engineered construct is added to cells which contain the targeted gene of interest. By mechanisms that are poorly understood but are similar to what occurrs during meiosis and mitosis when homolgous chromosomes align along the metaphase plane, the engineered

construct finds the targeted gene and recombination takes place within the homolgous (meaning identical in this case) sequences (figure 3). The recombination may take place anywhere within the flanking DNA sequences and the exact location is determined by the cells and not the investigators.

Figure 3. Diagram of alignment of DNA just prior to homolgous recombination. Amazingly, the DNA construct finds its way into the nucleus and then aligns itself with the targeted locus. The mechanism that performs this alignment is poorly understood but it does work better in some species than others. One of the best species for performing homologous recombination is yeast. Mouse is good mammalian system but homologous recombination does not work as efficeintly in human cells.

Once the cells have performed their part of the procedure, the end result is a new piece of DNA inserted into the chromosome. The rest of the genome is unaltered but the single targeted locus has been replaced with the engineered construct and some of its flanking DNA (figure 4). The original engineered construct has taken up the targeted gene of interest but since it cannot replicate in a nucleus, it is lost quickly in dividing cells while the modified chromosome replicates faithfully, including its new insert. Cells that have undergone homologous recombination can be selected by addition of antibiotic to the growth medium (positive selection). Notice that the negative selection marker is not incorporated into the chromosome by homologous recombination.

Figure 4. Diagram of the final products of homologous recombination. The chromosome now contains a portion of the flanking DNA as well as all of the engineered construct which has taken the place of the original allele. The original allele has been recombined into the construct and thus is lost over time. From this point on, the cell will replicate the engineered construct as faithfully as any other portion of the chromosome.

If the targeting vector aligns in a non-homologous region of the genome, then recombination is random and the negative selection marker may become incorporated into the genome (figure 5).

Figure 5. Alignment of DNA just prior to non-homolgous recombination. In this case, the recombination will occur randomly. Notice that this time the tk marker will be included in the recombination event.

The final product of non-homologous recombination (figure 6) can survive positive (antibiotic) selection. However, there is a drug called gancyclovir that will kill any cell that contains the tk gene. So cells undergoing homologous recombination are grown in antibiotic to select for recombination and gancyclovir to kill any cells that successfully conducted non-homologous recombination.

Figure 6. End products of non-homolgous recombination. The positive and negative selection markers are incorporated into the chromosome so gancyclovir will kill cells with modified chromosomes as shown here.

Knockout Mouse A knockout mouse has had both alleles of a particular gene replaced with an inactive allele. This is usually accomplished by using homologous recombination to replace one allele followed by two or more generations of selective breeding until an breeing pair are isolated that have both alleles of the targeted gene inactivaated or knocked out. Knock out mice allow investigators determine the role of a particular gene by observing the phenotype of individuals that lack the gene completely. Step 1. Isolate developing embryo at blastocyst stage. This embryo is from a strain of mice with gray fur.

Step 2. Remove embryonic stem cells from gray-fur blastocyst. Grow stem cells in tissue culture.

Step 3. Transfect stem cells with homologous recombination construct. Select for homologous recombination by growing stem cells in neomycin and gancyclvir.

+ + neomycin gancyclovir

Step 4. Remove homologously recombined stem cells from petri dish and inject into a new blastocyst that would have only white fur.

Step 5. Implant several chimeric blastocysts into pseudo-pregnant, white fur mouse.

Step 6. Mother will give birth to a range of mice. Some will be normal white fur mice but others will be chimeric mice. Chimeric mice have many of their cells from the original white fur blastocyst but some of their cells will be derived from recombinant stem cells. Fur cells from recombinant stem cells produce gray patches which are easily detected.

Step 7. Mate the chimeric mice with wild-type white fur mice. If the gonads of the chimeric mice were derived from recombinant stem cells, all the offspring will have gray fur. Every cell in gray mice are heterozygous for the homologous recombination.

Step 8. Mate heterozygous gray mice (+/ H) and genotpye the gray offspring. Identify homozygous recombinants (H / H) and breed them to produce a strain of mice with both alleles knocked out. The pure breeding mouse strain is a "knockout mouse".

knockout mouse

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