Method for inhibiting expression of a protein in a hepatocyte
A method of screening a candidate compound for susceptibility to biliary excretion by a hepatocyte transport protein. In some embodiments the method can comprise inhibiting expression of the transport protein. Expression of the transport protein can be inhibited through introduction of a RNA having a sequence corresponding to a coding strand of the gene encoding the transport protein into the hepatocyte.
1. A method of inhibiting expression of a transport protein in a hepatocyte, the method comprising:
(a) providing a hepatocyte expressing a transport protein, wherein the transport protein is organic anion transporting polypeptide 1B1 (OATP1B1); and
(b) introducing an oligonucleotide into the hepatocyte in an amount sufficient to inhibit expression of the transport protein, wherein the oligonucleotide comprises a nucleotide sequence which corresponds to a coding strand of a gene encoding the transport protein.
2. The method of claim 1 , wherein the hepatocyte is present in an organism, and the oligonucleotide is introduced into the organism.
3. The method of claim 2 , wherein the organism is a mammal.
4. The method of claim 1 , wherein the oligonucleotide is a ribonucleic acid (RNA).
5. The method of claim 4 , further comprising introducing a vector into the hepatocyte, wherein the vector encodes the RNA.
6. The method of claim 1 , wherein the hepatocyte is present in a culture.
7. The method of claim 1 , wherein the hepatocyte is isolated from a source selected from the group consisting of rat, human, monkey, ape, cat, dog, pig, hog, cattle, oxen, sheep, horses, turkeys, chickens, ducks and geese.
8. The method of claim 6 , wherein the culture of hepatocytes further comprises a long-term culture of hepatocytes.
9. The method of claim 6 , wherein the culture of hepatocytes further comprises a canalicular network.
10. The method of claim 6 , wherein the culture of hepatocytes is further characterized as having a configuration selected from the group consisting of clusters of hepatocytes, aggregates of hepatocytes, at least one layer of hepatocytes, and combinations thereof.
11. The method of claim 10 , wherein the hepatocytes are embedded in a matrix.
12. The method of claim 10 , wherein the culture of hepatocytes further comprises a sandwich culture of hepatocytes, the sandwich culture comprising at least one layer of hepatocytes and at least one bile canaliculus with the at least one layer of hepatocytes.
13. The method of claim 12 , wherein the sandwich culture of hepatocytes further comprises a long-term sandwich culture of hepatocytes.
14. The method of claim 12 , wherein the at least one layer of hepatocytes is sandwiched between two layers of matrix.
15. The method of claim 14 , wherein the matrix is selected from the group consisting of a biological matrix medium, a synthetic matrix medium, and combinations thereof.
16. The method of claim 14 , wherein the biological matrix medium is selected from the group consisting of collagens, laminins, basement membrane-derived complexes, derivatives thereof and combinations thereof.
17. The method of claim 1 , wherein the transport protein is a human transport protein.
18. The method of claim 1 , wherein the transport protein is a rodent transport protein.
19. The method of claim 17 , wherein the gene encodes OATP1B1.
20. The method of claim 4 , wherein the RNA comprises a double-stranded region comprising a first strand comprising a ribonucleotide sequence that corresponds to the coding strand of the gene encoding the hepatocyte transport protein and a second strand comprising a ribonucleotide sequence that is complementary to the first strand, and wherein the first strand and the second strand hybridize to each other to form the double-stranded molecule.
21. The method of claim 20 , wherein the double stranded region is at least 15 basepairs in length.
22. The method of claim 21 , wherein the double stranded region is between 15 and 50 basepairs in length.
23. The method of claim 22 , wherein the double stranded region is between 15 and 30 basepairs in length.
24. The method of claim 20 , wherein the RNA comprises one strand that forms a double-stranded region by intramolecular self-hybridization.
25. The method of claim 24 , wherein the double-stranded region is complementary over at least 19 bases.
26. The method of claim 20 , wherein the RNA comprises two separate strands that form a double-stranded region by intermolecular hybridization.
27. The method of claim 26 , wherein the double-stranded region is complementary over at least 19 bases.
28. The method of claim 1 , wherein the expression of the gene encoding the protein is inhibited by at least 10%.
29. The method of claim 1 , wherein introducing the oligonucleotide into the hepatocyte comprises transfecting the oligonucleotide into the hepatocyte.