Generation of the HRN mouse

In order to understand the contribution of P450 metabolism to the in vivo disposition of a compound it is useful to determine the pharmacokinetics in the absence of drug metabolism. The size and diversity of the P450 multigene family makes it impractical to either simultaneously inhibit all the enzyme activity, or knockout the numerous genes, and so an alternative strategy was required. All cytochrome P450s receive electrons from a single donor, cytochrome P450 reductase (CPR), so that deletion of this protein would inactivate all CYPs. Complete knock out of this gene was however shown to be embryonically lethal, demonstrating that P450 activity is essential for normal development, and that a different deletion strategy was required. This was provided by the Cre/loxP system, which was used to induce conditional tissue specific postnatal deletion of CPR. A mouse line was generated that contained loxP sites inserted on either side of the CPR gene (figure1).

Figure 1: Targeting of the mouse CPR gene.Maps of the wild-type, floxed and disrupted CPR alleles. The CPR gene is indicated by squares 5-15 and LoxP sites are indicated by triangles. The activity of Cre recombinase, recombines both LoxP sites and so excises out the CPR gene.

This line was bred with a transgenic mouse line that expressed the bacteriophage Cre recombinase under control of the albumin promoter. As Cre catalyses the recombination of the loxP sites to excise the CPR gene, and as albumin is only expressed postnatally in the liver, this restricts the deletion of CPR to the adult liver. Adult offspring of this pairing therefore lack hepatic CPR but retain normal CPR expression, and hence P450 activity, in other tissues (see figure 2).

Figure 2: Cytochrome P450 reductase activity and CPR levels in liver microsomes from HRN™ mice relative to controls. Immunochemical detection of cytochrome P450 reductase by western blot analysis: lanes 2 - 5 are controls, and lane 1 is HRN, for both male and female mice. Units of CPR activity: µmoles cytochrome C reduced/min/mg microsomal protein. Cytochrome P450 reductase is undetectable in liver microsomes by western blotting (bottom panel) and this correlates with attenuation of CPR activity (top panel).