The usual approach towards the study of gene function is to insert or inactivate the gene in a cell or an individual, and to observe changes in cell biological behavior or individual phenotypes to identify its function.
Therefore the study of gene function can be conducted following the following two types of strategies:
Loss of function
Gene knockout is one of the most frequently-used methods to study loss of function in mice. Gene knockout refers to the discovery of biological functions of target genes by studying organisms which carry the inactivated (knocked out) target genes. This technique requires changes to be made in partial sequences of target genes, which disable the functions of the specific genes and thereby inactivating part or all of the target gene function. Gene knockout can be further divided into conventional gene knockout and conditional gene knockout.
Conventional Gene Knockout
Conventional Knockout (abbreviation: KO) refers to the knockout of certain vital exons, functional domains of the target gene, or even all exons, in all mouse cells, thereby resulting in loss of expression of the target gene.
KO mice carry the engineered target gene sequences in all tissues and cells. Target genes are not expressed in homozygous mice
KO mice are generally used to study the effects of target genes or protein functions on physiology or pathology.
Conditional Gene knockout
Conditional Knockout (abbreviation: CKO) is the temporal and spatial-specific engineering of the mouse genome by limiting gene engineering to certain specific types of cells in the mouse or to a specific stage of development
Gene targeting is used in CKO mice to flank one or several important exons of the target gene with loxP sites. Expression of the target gene was normal before crossing with the Cre recombinase-expressing strain. When bred with Cre recombinase-expressing transgenic mice, the target gene can be knocked out in a specific tissue or cell type, and its expression will remain normal in other tissues or cells types.
CKO mice are generally used to study genes that cause embryonic lethality, functions of target genes or proteins in a particular tissue or cell type, or the role of target genes or proteins at a particular period or stage.
Gain of function
The study of gene function by observing changes in biological traits of the cell or individual, in which the target gene had been introduced to generate new or higher levels of expression.
Random insertion transgenesis is the random integration of an exogenous gene into the mouse genome to generate mouse models with overexpression of the target gene. It is used to study the impacts of target gene overexpression on physiology and pathology.
Targeted gene overexpression
The exogenous DNA sequence is stably expressed in mice after being knocked into the mouse Gt (ROSA) 26Sor also known as Rosa26 gene locus. Expression of the exogenous gene can also be tissue-specific or drug-induced.
Useful for overexpression experiments on gene and protein function
Can be used for rescue experiments for KO phenotypes
Knock-in (abbreviation: KI) refers to the introduction of specific mutations or exogenous DNA sequences at the site of the target gene.
(1) Introduction of a base mutation into the target gene to mimic a human genetic disease model.
(2) Introduction of a reporter gene, such as EGFP, RFP, mCherry, YFP, LacZ, and Luciferase, into the murine endogenous gene to observe the expression pattern of the target gene.