Introduction

Genes are the fundamental units of heredity that carry the information necessary for the development and function of all living organisms. The study of gene function is essential for understanding the molecular mechanisms underlying various biological processes. One of the most powerful tools for studying gene function is the ability to disrupt or reduce the expression of specific genes. The two most widely used methods for this are the knock out and knock down mechanisms. This chapter will discuss the molecular basis of these mechanisms. These include the methods used to generate knock outs and knock downs, the advantages and disadvantages of each, and the downstream effects of gene disruption.

Knock Out Mechanism

A knock out is a genetic manipulation in which a specific gene is inactivated or “knocked out” by disrupting its function. This is typically achieved by introducing a mutation into the gene that disrupts its coding sequence, rendering it non-functional. The most common method for generating a knock out is homologous recombination, in which a DNA construct containing the mutation is introduced into cells. Then the cells are selected for the presence of the mutation. This method can be used to generate knock outs in both prokaryotic and eukaryotic organisms. And it is the most widely used method for generating knock outs in mice.

Knock Down Mechanism

A knock down is a genetic manipulation in which the expression of a specific gene is reduced. This is typically achieved by introducing small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) into cells. These siRNAs bind to and degrade specific mRNA molecules, thereby reducing the amount of protein produced. RNA interference (RNAi) is the most common method for generating a knock down. It is widely used in both prokaryotic and eukaryotic organisms.

Advantages and Disadvantages

Knock out and knock down mechanisms have their own advantages and disadvantages. Knock outs are permanent, meaning the gene is inactivated for the entire lifetime of the organism. This allows for the study of the long-term effects of gene disruption. However, knock outs can also lead to pleiotropic effects, meaning that disrupting one gene can have unintended effects on other genes or pathways. In contrast, knock downs are reversible, meaning the gene expression can be restored once the siRNA or shRNA is removed. This allows for the study of the short-term effects of gene disruption. However, knock downs can also lead to off-target effects. It means that the siRNA or shRNA can bind to and reduce the expression of other genes that share similar sequences.

Downstream Effects

The downstream effects of gene disruption can vary depending on the gene and the organism being studied. In general, knock outs can lead to the complete absence of a protein, whereas knock downs can lead to a reduction in the amount of protein produced. This can result in a wide range of effects. Those include changes in cell growth and division, changes in protein interactions, and changes in the regulation of other genes.

Conclusion

The molecular basis of knock out and knock down mechanisms of gene is a powerful tool for studying gene function. The knock out mechanism allows for the study of the long-term effects of gene disruption. The knock down mechanism allows for the study of the short-term effects. Both methods have their advantages and disadvantages, and the downstream effects of gene disruption can vary depending on the gene and the organism being studied. Understanding the molecular basis of these mechanisms is essential for understanding the molecular mechanisms underlying various biological processes.