Gene Knockout

Gene Knockout (KO) is a technique by which the genomic DNA of a cell or a model organism is perturbed, so that the expression of a specific gene is permanently prevented. If the cells or model organisms survive a knockout event, they can never return to expressing the functional gene product.

Gene knockout does not mean that an entire gene must be physically excised or cut out from the genome. Instead, when a gene is knocked out, a frameshift mutation leading to a stop codon is usually introduced near the 5' end of the gene (near the start site of transcription). All transcription downstream of the stop codon is permanently prevented, so the gene is knocked out.

Process

• Selecting a gene for knockout
  Select a gene that we want to study or whose function we wish to understand.
• Construction of vector
  Vector is a vehicle used to transfer our gene of interest or any other DNA sequence to our target cells, a plasmid is generally used for it. A marker DNA sequence is also introduced in it to validate the results, generally, an antibiotic resistance gene is used for it.
• Insertion into ES cells
  Using artificial methods such as electroporation, sonication or microinjection, the plasmid is inserted into the ES cells. The electrophoration method is one of the best techniques used in gene knockout in which under the electrical current, a gene is inserted into the cell.
• Confirming the insert
  Using the polymerase chain reaction, the insert can be confirmed. For that, DNA is extracted from the cultured cells. A set of primers specific to our marker DNA sequence is used for achieving amplification. If the amplicons are observed, cells are transformed otherwise our experiment is failed.
• Injecting into the embryo
  Pick transformed cells and insert them into the developing embryo of the model organism. Let it grow normally.
• Breeding
  Now the embryo of our model organism has two types of cell population, one wild type and one altered (transformed) cell, this animal is called chimeric.
• Examination
  Gene knockout animal is constructed. Examine it for measuring a different parameters related to our gene of interest such as physical, biochemical or genetic parameters.

Fig.1 The entire process of gene knockout.

Methods

• Gene silencing
  Gene silencing is a modern gene-editing technique used for genetic engineering experiments. Using techniques like RNA interference, CRISPR-CAS9 and antisense RNA technique, a gene of our interest can be suppressed or its expression is controlled.
• Conditional knockout
  The conditional knockout method is used to inactivate the gene in a specific tissue at a specific time for a specific function. It is done in the adult animal instead of during the embryonic stage through the process of homologous recombination.
• Homologous recombination
  One of the traditional and widely used methods for studying gene knockout in genetic engineering is homologous recombination. Exchange of the nucleic acid between identical or homologous sequences occurs through homologous recombination.
• Gene editing
  Gene editing is one of the emerging tools in recent day genetics where scientists are using nucleases to remove nucleic acid using homologous recombination. ZFNs, TALENs and CRISPR-CS9 are gene-editing nucleases used in genetic engineering.
• Knockout by mutation
  A loss of function mutation can help to suppress gene function by creating a mutation. Either using chemical mutagens or physical agents, scientists can introduce mutation in a gene, however, the specificity and accuracy are very low.

Applications

• Study the function of a particular gene
• Monitor and control the effect of a gene
• For constructing genetically modified organisms such as GM plants, GM bacteria and GM animals
• Study the effect and contribution of a particular gene and its role in the development of a disease
• In drug discovery: using gene knockout like genetic engineering tools, drug screening can be done

Related Products

Flow Cytometry

Flow Cytometry is a sophisticated instrument measuring multiple physical characteristics of a single cell such as size and granularity simultaneously as the cell flows in suspension through a measuring device. Its working depends on the light scattering features of the cells under investigation, which may be derived from dyes or monoclonal antibodies targeting either extracellular molecules located on the surface or intracellular molecules inside the cell.

Centrifuge

A centrifuge is any device that applies a sustained centrifugal force—that is, a force due to rotation. The widest use of centrifuges is for the concentration and purification of materials in suspension or dissolved in fluids. Suspended particles denser than the suspending liquid tend to migrate toward the periphery, while those less dense move toward the centre.

Electroporation

Electroporation and transfection instruments enable the introduction of exogenous nucleic acids or other molecules into cells. Electroporation systems, sometimes called electroporators, send out brief electric pulses to disrupt plasma membranes and allow entry of DNA or RNA. These instruments are designed for a broad spectrum of eukaryotic and prokaryotic cell types, including microbial, plant, primary cells, and stem cells.

Digital PCR System (dPCR)

Digital PCR Systems (dPCR) is incredibly useful in applications such as mutation detection, copy number variation, rare sequence detection, gene expression analysis, miRNA analysis and next generation sequencing sample quantification. It provides a precise measure of DNA molecules in each drop. In some cases, Digital PCR Systems (dPCR) must be used in combination with a 96-deep well reaction module.

Laboratory Incubator

An incubator is a device used to grow and maintain microbiological cultures or cell cultures. It is made up of a chamber with a regulated temperature. An incubator maintains optimal temperature, humidity, and other conditions such as the CO2 and oxygen content of the atmosphere inside. Incubators are essential for much experimental work in cell biology, microbiology, and molecular biology.

Pipette

Pipette is a small piece of apparatus which typically consists of a narrow tube into which fluid is drawn by suction (as for dispensing or measurement) and retained by closing the upper end. Pipettes are a common laboratory tool and come in a multitude of channels, displacements and construction materials. There are different pipettes for all kinds of uses within the lab.

STEMart provides you with a variety of gene knockout equipment or consumables to meet your various R&D and application needs. If you have any questions or requirements for techniques for the study of gene function, please feel free to contact us.