Targeting strategy
Gene targeting strategy for B-Mdr1 KO mice. 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.
mRNA expression analysis
Mdr1a mRNA expression analysis in wild-type C57BL/6 mice and B-Mdr1 KO mice by RT-PCR. Brain, lung and liver RNA were isolated from wild-type C57BL/6 mice and B-Mdr1 KO mice, then cDNA libraries were synthesized by reverse transcription, followed by PCR with two mouse Mdr1a primer pairs. Mouse Mdr1a mRNA was not detectable in Mdr-1 knock-out mice.
Mdr1b mRNA expression analysis in wild-type C57BL/6 mice and B-Mdr1 KO mice by RT-PCR. Brain, lung and liver RNA were isolated from wild-type C57BL/6 mice and B-Mdr1 KO mice, then cDNA libraries were synthesized by reverse transcription, followed by PCR with two mouse Mdr1b primer pairs. Mouse Mdr1b mRNA was not detectable in Mdr-1 knock-out mice.
Protein expression analysis
Western blot analysis of MDR1 protein expression in B-Mdr1 KO mice. Various tissue lysates were collected from wild-type C57BL/6 mice and B-Mdr1 KO mice, and then analyzed by western blot with anti-MDR1A antibody. 40μg total proteins were loaded for western blotting analysis. MDR1 was only detectable in adrenal, brain, kidney and lung from wild-type mice but not in Mdr-1 knock-out mice.
