The Knockout Mice Core offers six vector systems to scientists interested in determining the role of a particular gene.
This is a general knock out replacing exon 2 with a neomycin resistance cassette. This most basic of the systems works as well as any for a simple knock out.
In this knock out we have flanked the Neomycin cassette with Lox P site. This will allow either in vitro or in vivo removal of the Neo cassette. This is to remove any potential effects of the neo promoter, which is generally a strong ‘bi-directional’ system.
Similar to the Lox-Neo construct, this vector has Lox sites flanking the neo cassette. The difference is that one of the sites is a mutated Lox P, a Lox 71. This mutation will allow for the insertion is a plasmid in the future to either replace the removed exon with a modified version (see Knock In) or any other DNA you choose.
Conditional Knock Out
In this most useful of knock outs, the exon of choice is flanked by the Lox P sites, This will allow the excision of the inclusive region by use of any of hundreds of tissue and stage specific Cre transgenes. A great choice when dealing with lethal knockouts and broad ranging phenotypes. The Neo cassette in this construct is flanked by FRT sites, this will allow for the removal of the neo, with out affecting the rest of the gene.
Reporter Knock Out
This ‘two tools in one’ approach gives the investigator both a basic knock out, and lacZ reporter knock in. Having the same effect of removing the exon of choice, it will act as a knock out mouse, but the addition of the LacZ (or any other) reporter gene will reveal the expression pattern of the gene of interest.
The Knock in could prove to be one of the more powerful systems we have. Two of the more interesting uses would be the introduction of point (or small) mutations, and the humanized mouse model. The introduction of the point mutation can generate either a complete loss of function, or a partial loss of function. In the humanized model, genes or exons can be replaced with their human equivalent when there seems to be a functional difference.