Targeting strategy

Gene targeting strategy for B-Mdr1/Brcp KO mice. The exons 2~16 of mouse Bcrp gene were knocked out in B-Bcrp KO mice, resulting in a disruption of the Bcrp gene.

The genome sequences between mouse Mdr1a gene and Mdr1b gene were depleted in B-Mdr1 KO mice. As a result, mouse MDR1 protein will be not expressed anymore.

B-Mdr1/Brcp KO mice were obtained by mating B-Mdr1 KO mice and B-Bcrp KO mice.

mRNA expression analysis

mRNA expression analysis in wild-type C57BL/6 mice and B-Mdr1/Bcrp KO mice by RT-PCR. Brain and liver RNA were isolated from wild-type C57BL/6 mice and B-Mdr1/Bcrp KO mice, then cDNA libraries were synthesized by reverse transcription, followed by PCR with  mouse Mdr1a, Mdr1b and Bcrp primer pairs. Mouse Mdr1a, mouse Mdr1b and mouse Bcrp mRNA were not detectable in B-Mdr1/Bcrp KO mice.

Protein expression analysis

Western blot analysis of MDR1 and BCRP protein expression in B-Mdr1/Bcrp KO mice. Various tissue lysates were collected from wild-type C57BL/6 mice and B-Mdr1/Bcrp KO mice, and then analyzed by western blot with anti-MDR1 antibody (abcam, ab170904) and anti-BCRP antibody (abcam, ab207732). 40μg total proteins were loaded for western blotting analysis. MDR1 was only detectable in brain, lung, large intestine and stomach from wild-type mice but not in B-Mdr1/Bcrp KO mice, BCRP was only detectable in kidney, liver, large intestine from wild-type mice but not in B-Mdr1/Bcrp KO mice.