Introduction:

A “knockout” animal has had a gene inactivated so that the funciton of the gene is lost. It is possible to inactivate genes in a number of different experimental organisms. The most relevant to human genetics is the mouse.

There are a couple shortcoming to knocking out genes in mice. First, a gene knockout results in all cells of all tissues being affected bys the inactivation. This is problematic for genes whose products affect more than one tissue. The phenotypic analysis of a knockout that affects mutiple tissues can be complex and may not even be possible. Second, there are developmentally important genes. A gene that is essential for early developmetn will lead to embryonic lethality, and may leave nothing to anlayze. There are however ays such as the Cre-Lox recombination system which allow for conditional inactivation so that a gene to be kncoked out in cells of specific tissue or at specific time in development can be accomplished. Cre is an enzyme that catalyzes recobmination between short DNA sequences called Lox sequences. If a gene is flancked by Lox sites, then in the presence of the Cre enyme, recombination between the Lox sites will remove the itnervening sequence of DNA (the gene of interest) as a circular DNA that will be degrated. The promoter expressing Cre is turned on in the presence of an inducing molecule, or in a particular tissue. 

There are several difficulties in using knockout mice in providing conclusive evidence for genes in disease traits, like the role of OPN in inflammatory diseases. A common pitfall is when wild-type mice and not littermates are used as control. In that situtation, any gene in the genome that differs between the embroyonic stem 9ES) cell and the back-crossed parental cell many play a role. Preferably, heterozygous littermates should be included, because dominant genes in the linked fragment will be neutralized. However, a recessive effect closely linked to the gene of interest is almost impossible to exclude if the knockout is created with ES cells of a different genotype than in the mouse used later in the study (“Comment on the “The influence of the proinflammatory cytokine, osteopontin, on autoimmune demyelinating disease”, Science, 299, March 2003).

Procedure:

Some of the stps used in creating a knowout mosue include the follwoing:

Disruption of a clone gene by replacing part of it with a marker gene which codes for resistance to the antibiotic neomysin in bacteria, which allows mosue cells to survive when grown in a medium containing the related rug G418. The construction is done such that the marker gene is flanked by the DNA normally flanking the gene of interest in the chromosome.

Introduction into embryonic stem cells derived form early embryos which can develop into different aadult tissues. In these cells, the gene can recombine with the chromosomal copy of the gene based on the flanking DNA. The knockout gene with the drug resitance gene does not have an origin of replicaiton, and thus it will be lsot if no recombination occurs. Cells are grown in medium containing G418 to select for recombination. 

Injuction of the ES cells containing the knocked-out allele into a mouse embryo early in its devleopment, which is then iimplanted into a pseudopregant famle (a femal that has been materd with a vascetomized male and as a result has a receptive uterus) Some of the cells in the pups born to this female have one of the two alleles for the gene of interest knocked out and so these animals are hcimeras. A chimeric mouse is maated with a normal mosue and some of the offspring will be completely heroxygous. In some cases there may be a phenotype that can be detected and anlyzed if heterozgosity affects any biologcial funciton. If the mutation is recessive and not lethal, heterozygotes can then be crossed to generate homozygous mice which can then be analyzed for phenotypes.