An endogenous gene has been deleted resulting in a loss of protein expression from that gene
Usual purpose is to study what happens when a protein product is missing (e.g., to mimic a human disease condition)
One disadvantage is compensatory actions of homologous genes may mask some of the phenotype
Gene added - either a foreign gene (e.g., fluorescent protein or an endogenous gene driven by an independent promoter and located on a different genetic locus (sometimes referred to as overexpression or misexpression mice))
Purpose – to manipulate genome to gain insight into a cellular process or protein function or to create new functions to help understand physiological responses
Disadvantage - there may be little control over expression levels both spatially and temporally
Gene targeting - What makes an arm an arm
Capecchi's research team has systematically "knocked out" a set of genes in mice, called homeotic genes, which govern body patterning during development
How you Make a Transgenic Mouse
Isolate embryonic stem cells that originated from male brown mice with a normal OhNo gene (blue)
Add Inactive Gene With Marker - To these cells, add a copy containing a mutated, inactive OhNo gene (red), and a drug resistance marker gene (pink)
Similar genes will swap places - The OhNo gene plus drug resistance marker gene is incorporated into the genome, and the normal version is kicked out (homologous recombination)
Adding the drug kills cells without the marker, leaving you with only cells that have an inactive version of the OhNo gene.
Transplanted stem cells carrying the inactive OhNo gene in a white mouse embyro creates a chimera (have patches of white mouse cells and patches of brown stem cells (have both brown and white patches of fur))
½ brown offspring will have copy of inactive OhNo gene in all of their cells—including reproductive cells. These mice have one normal copy of the OhNo gene from their mother (not shown) and one inactive copy from their father. These mice can be identified by performing DNA sequencing in their OhNo genes and then bred with each other.
One fourth of your resulting offsping will have two copies of the "knocked-out" or inactive OhNo gene