Construction of HEK293 cells stably expressing wild-type organic anion transporting polypeptide 1B1 (OATP1B1*1a) and variant OATP1B1*1b and OATP1B1*15
A transgenic cell line stably expressing the human organic anion transporting polypeptide (OATP1B1) was established. Human Embryonic Kidney 293 (HEK293) cell line stably expressing OATP1B1*1a sequence was amplified through PCR with the extracted total RNA as templates from human liver, then subcloned into the plasmid pMD19-T and verified by sequencing. OATP1B1*1b/OATP1B1*15 mutant sequences were obtained by site-directed mutation PCR with pMD19-T/ OATP1B1*1a as templates. The plasmids pcDNA3.1(+)/OATP1B1*1a, *1b and *15 were constructed and transfected into HEK293 cell line using Lipofectamine 2000 transfection reagent. Several stable transfected clones were obtained after selection with G418. Using rosuvastatin as a probe substrate of OATP1B1, the intracellular rosuvastatin accumulation in HEK293 and HEK-OATP1B1*1a, *1b and *15 monoclone cells were validated by a ultra-performance liquid chromatography-tandem mass spectrometry. OATP1B1 mRNA and protein expression were detected by RT-PCR and Western blot, respectively. The results from RT-PCR, rosuvastatin uptake and Western blot assay indicated that human OATP1B1 was highly expressed in transfected cells compared with controls. The HEK-293 cell lines stably expressing human OATP1B1-wild and variant (HEK-OATP1B1, *1b and *15) are potential models to study drug transport in vitro.
| Medienart: |
Artikel |
|---|
| Erscheinungsjahr: |
2016 |
|---|---|
| Erschienen: |
2016 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:71 |
|---|---|
| Enthalten in: |
Die Pharmazie - 71(2016), 6 vom: 01. Juni, Seite 337-9 |
| Sprache: |
Englisch |
|---|
| Beteiligte Personen: |
Chen, M [VerfasserIn] |
|---|
| Anmerkungen: |
Date Completed 17.08.2016 Date Revised 02.12.2018 published: Print Citation Status MEDLINE |
|---|
| Förderinstitution / Projekttitel: |
|
|---|
| PPN (Katalog-ID): |
NLM262783037 |
|---|
| LEADER | 01000naa a22002652 4500 | ||
|---|---|---|---|
| 001 | NLM262783037 | ||
| 003 | DE-627 | ||
| 005 | 20231224202503.0 | ||
| 007 | tu | ||
| 008 | 231224s2016 xx ||||| 00| ||eng c | ||
| 028 | 5 | 2 | |a pubmed24n0875.xml |
| 035 | |a (DE-627)NLM262783037 | ||
| 035 | |a (NLM)27455553 | ||
| 040 | |a DE-627 |b ger |c DE-627 |e rakwb | ||
| 041 | |a eng | ||
| 100 | 1 | |a Chen, M |e verfasserin |4 aut | |
| 245 | 1 | 0 | |a Construction of HEK293 cells stably expressing wild-type organic anion transporting polypeptide 1B1 (OATP1B1*1a) and variant OATP1B1*1b and OATP1B1*15 |
| 264 | 1 | |c 2016 | |
| 336 | |a Text |b txt |2 rdacontent | ||
| 337 | |a ohne Hilfsmittel zu benutzen |b n |2 rdamedia | ||
| 338 | |a Band |b nc |2 rdacarrier | ||
| 500 | |a Date Completed 17.08.2016 | ||
| 500 | |a Date Revised 02.12.2018 | ||
| 500 | |a published: Print | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a A transgenic cell line stably expressing the human organic anion transporting polypeptide (OATP1B1) was established. Human Embryonic Kidney 293 (HEK293) cell line stably expressing OATP1B1*1a sequence was amplified through PCR with the extracted total RNA as templates from human liver, then subcloned into the plasmid pMD19-T and verified by sequencing. OATP1B1*1b/OATP1B1*15 mutant sequences were obtained by site-directed mutation PCR with pMD19-T/ OATP1B1*1a as templates. The plasmids pcDNA3.1(+)/OATP1B1*1a, *1b and *15 were constructed and transfected into HEK293 cell line using Lipofectamine 2000 transfection reagent. Several stable transfected clones were obtained after selection with G418. Using rosuvastatin as a probe substrate of OATP1B1, the intracellular rosuvastatin accumulation in HEK293 and HEK-OATP1B1*1a, *1b and *15 monoclone cells were validated by a ultra-performance liquid chromatography-tandem mass spectrometry. OATP1B1 mRNA and protein expression were detected by RT-PCR and Western blot, respectively. The results from RT-PCR, rosuvastatin uptake and Western blot assay indicated that human OATP1B1 was highly expressed in transfected cells compared with controls. The HEK-293 cell lines stably expressing human OATP1B1-wild and variant (HEK-OATP1B1, *1b and *15) are potential models to study drug transport in vitro | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Research Support, Non-U.S. Gov't | |
| 650 | 7 | |a Hydroxymethylglutaryl-CoA Reductase Inhibitors |2 NLM | |
| 650 | 7 | |a Lipids |2 NLM | |
| 650 | 7 | |a Lipofectamine |2 NLM | |
| 650 | 7 | |a Liver-Specific Organic Anion Transporter 1 |2 NLM | |
| 650 | 7 | |a Organic Anion Transporters |2 NLM | |
| 650 | 7 | |a RNA, Messenger |2 NLM | |
| 650 | 7 | |a SLCO1B1 protein, human |2 NLM | |
| 650 | 7 | |a Rosuvastatin Calcium |2 NLM | |
| 650 | 7 | |a 83MVU38M7Q |2 NLM | |
| 700 | 1 | |a Qu, B X |e verfasserin |4 aut | |
| 700 | 1 | |a Chen, X L |e verfasserin |4 aut | |
| 700 | 1 | |a Hu, H H |e verfasserin |4 aut | |
| 700 | 1 | |a Jiang, H D |e verfasserin |4 aut | |
| 700 | 1 | |a Yu, L S |e verfasserin |4 aut | |
| 700 | 1 | |a Zhou, Q |e verfasserin |4 aut | |
| 700 | 1 | |a Zeng, S |e verfasserin |4 aut | |
| 773 | 0 | 8 | |i Enthalten in |t Die Pharmazie |d 1947 |g 71(2016), 6 vom: 01. Juni, Seite 337-9 |w (DE-627)NLM000009180 |x 0031-7144 |7 nnns |
| 773 | 1 | 8 | |g volume:71 |g year:2016 |g number:6 |g day:01 |g month:06 |g pages:337-9 |
| 912 | |a GBV_USEFLAG_A | ||
| 912 | |a GBV_NLM | ||
| 951 | |a AR | ||
| 952 | |d 71 |j 2016 |e 6 |b 01 |c 06 |h 337-9 | ||