Reverse Genetics

This begins with the known sequence of a gene and techniques used include RNA interface and the creation of deletion mutants. These are used to study the effects on an organism when a gene is knocked down or removed. RNAi creates a knockdown of gene function, whereas a deletion mutation permanently removes the gene’s function.

To begin with a gene is cloned into a plasmid and sequenced, then through the use of reverse genetic techniques it can be analysed. This can involve the use of things such as morpholinos, mouse genetics and siRNA.

Reverse genetics is aimed at finding the possible phenotypes that may be derived from a specific genetic sequence, that is detailed in DNA sequencing. Making a change or disruption in the DNA can enable researchers to discover the influence a sequence has on a phenotype, or discover its biological function. This is done by making an alteration and then looking for the effects it has on the organism.

RNAi

RNA interface is often used in experimental biology to study the function of genes. It is a commonly used mechanism that inhibits gene expression, thus either hindering their transcription or preventing translation. This technique only causes a knockdown of gene function, and therefore doesn’t totally abolish the expression of the gene.

Double-stranded RNA complimentary to the gene  is introduced into a cell, and proceeds to activate the RNAi pathway. The dsRNA activates a ribonuclease which cleaves it into smaller fragments, which are known as small interfering RNA’s. These bind to the RNA-induced silencing complex (RISC) that uses one strand of the siRNA to base pair to their target mRNA. The activity of RISC causes mRNA to be cleaved so it can no longer function as a template for translation.  Thus knocking down the synthesis of protein.

Mouse Genetics

One of the most common techniques mice are used for is gene knockout. This is usually performed when the sequence of the gene is known, and is used to discover its function. By inactivating a gene and examining the consequences, the function of a gene can often be determined.

Mice are ideal model organisms for this because of their homology to humans, many genes are conserved between mice and humans. These sorts of experiments can help contribute to the knowledge of how genes may act in disease, and thus investigate potential treatments for them.

Gene knockout causes genetic engineering of an organism to carry genes that have been silenced in their function and are no longer present. Often a transgenic animal is created that carries the altered gene, and this is incorporated into the animals genome using recombinant DNA technology. A gene construct is inserted into a plasmid which transfects the animal, and the construct proceeds to recombine with the gene. This causes insertion of a foreign sequence into part of the gene which disrupts its sequence, so it can no longer be translated into a functional protein.