INTRODUCTION

The Secrets Behind Gene Knockout Technology

Presently, knockout gene technology or gene knockout technology (abbreviation: KO) allows experimenters to inactivate specific genes within an organism and determine the effect of this on the functioning of the organism. Also, they are used in learning about a gene that has been sequenced, but which has unknown or incompletely known function. Genes can be knocked out in microorganisms as well as organisms of greater complexity, such as mosquitoes, flies, and worms. However, the mouse is perhaps the most important species in which researchers can now use in this technology because of two reasons: (1) the mouse is the most genetically manipulated mammal in the world and (2) it shares much of its genetic material with humans (and other mammals) which means that experimental findings in mice will often be directly relevant to conditions in humans.

The paper focuses on the controversies and challenges in Gene Knockout Technology – an article/blog entitled “Sickle Cell Disease cured by Gene Knock-out”.

Switching off a single gene can help treat sickle cell disease or sickle cell anemia (a chronic, usually fatal anemia marked by sickle-shaped red blood cells, occurring almost exclusively in Black people of Africa or of African descent, and characterized by episodic pain in the joints, fever, leg ulcers, and jaundice. The disease occurs in individuals who are homozygous for a mutant hemoglobin gene) by keeping the blood forever young. The illness is caused by a mutant form of adult haemoglobin, but not by fetal haemoglobin. Targeting BCL11A, the gene responsible for the body's switch-over from fetal to adult haemoglobin, effectively eliminates the condition in mice.
The mutant form of adult haemoglobin forms long sticky chains inside red blood cells. The cells containing these chains can clog small blood vessels, depriving organs of oxygen and causing pain. In severe cases, sickle cell disease can be fatal. Tricking the body into make fetal haemoglobin again can alleviate symptoms, though.
That's because fetal haemoglobin does not form sticky chains. However, it is produced in the body only during development in the womb and in the six months following birth. It has a higher affinity for oxygen than adult haemoglobin, vital in allowing the developing fetus to "steal" oxygen from its mother's blood.
Stuart Orkin of Harvard Medical School in Boston, and colleagues, knocked out the BCL11A gene from mice belonging to a strain that normally develops a sickle cell-like condition. As adults, the mice produced over 20 times more fetal haemoglobin than normal and their blood contained almost no sickle-shaped cells. Their spleen and kidneys – organs easily damaged by the effects of the disease – were almost completely healthy.

Bound and gagged
Gene therapy to block the action of BCL11A in humans could in theory provide similar benefits. Specially designed lengths of RNA, injected into the bloodstream, could bind with the BCL11A gene and silence it. This approach, however, would be expensive and impractical on a large scale. "The long-term goal is to have a drug that can effectively block the function of BCL11A," says Orkin. "It's a more challenging approach, but one that could be applied to large populations."
Other treatments designed to encourage the production of fetal haemoglobin have been suggested over the years. One drug in particular – hydroxyurea – is widely used but has many side effects including reducing the levels of white blood cells.
Orkin's main concern with hydroxyurea, however, is that its effect on the body is not fully understood. "We have no idea how it really works. In some patients it's good, in others it doesn't work at all. It's unpredictable," he says. "Our approach gets to the real mechanism. It actually silences the gene that leads to fetal haemoglobin being suppressed."

Sickle Cell Anemia (a closer view) Hydroxyurea 500 mg capsule

SUMMARY
Definition
In biotechnology a knockout refers to an organism which has been genetically altered in such a way that one of its genes is missing or inactive due to a mutation, or its complete deletion. Since the discovery of genes, scientists have been determined to learn the structure and function of all their protein products. The construction of knockouts is one technique for doing this.

Methods Genes are knocked out by changing the region of the gene that codes for the protein and this is done by cloning the gene, manipulating it in bacteria, and injecting it into the nuclei of embryonic stem cells which are then placed in the developing test organisms. 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. 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.
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.

A knockout mouse (left) that is Wild-type Physcomitrella and knockout- a model of obesity, compared mosses. with a normal mouse. Significance in Exploring Physiologic Functions and Producing Animal Models for Human Disease The utilities for production of gene knockout mice are many-fold. First, gene knockout mice can be produced as an animal model for human diseases. These gene knockout mice can be used to explore potential intervention strategy, including pharmaceutical and genetic therapy approaches, for treatment of specific metabolic diseases. A second utility for the production of knockout mice is to explore physiological function and significance of specific genes.

Significance to Plant Metabolism Recent experience shows that knockouts of genes encoding enzymes of primary metabolism can produce mutants with clear and sometimes unexpected phenotypes. They can provide new information about old pathways. Specific functions for individual members of multigene families can be revealed. Knockouts of enzymes of undefined function can lead to the discovery of those functions, and the analysis of enzymes which have previously never been studied at the biochemical level offers the potential to reveal new pathways of plant metabolism. Furthermore, the mutants isolated provide the starting point for genetic modification experiments to determine exactly how metabolism fuels growth and development, so providing a rational basis for the future modification of plant productivity.

Limitations While knockout ice technology represents a valuable research tool, some important limitations exist. About 15 percent of gene knockouts are developmentally lethal, which means that the genetically altered embryos cannot grow into adult mice. The lack of adult mice limits studies to embryonic development and often makes it more difficult to determine a gene's function in relation to human health. In some instances, the gene may serve a different function in adults than in developing embryos.

Knocking out a gene also may fail to produce an observable change in a mouse or may even produce different characteristics from those observed in humans in which the same gene is inactivated. For example, mutations in the p53 gene are associated with more than half of human cancers and often lead to tumours in particular set of tissues. However, when the p53 gene is knocked out in mice, the animals develop tumours in a different array of tissues.

There is variability in the whole procedure depending largely on the strain from which the stem cells have been derived. Generally cells derived from 129 are used. This specific strain is not suitable for many experiments (e.g., behavioural), so it is very common to backcross the offspring to another strains. Some genomic loci have been proven very difficult to knock out. Reasons might be the presence of repetitive sequences, extensive DNA methylation, or heterochromatin.

CONCLUSION

Gene knockout technology is a wonderful tool used to study the function of a specific gene in a system. Despite of the numerous concerns and limitations of this technique, it provides tremendous insight into understanding the process of creating a knockout organism. The development of a gene knockout mouse has been a massive advance to the biomedical and pharmaceutical field presenting researchers with a very powerful tool for analyzing gene function during development, as well as in disease.