Technical Reference #1893

1893.

Heterologous functional expression system for odorant receptors Hiroshi Hamana; Li Shou-xin; Laure Breuils; Junzo Hirono; Takaaki Sato, National Institute of Advanced Industrial Science and Technology, Journal of Neuroscience Methods, 185(1893), (2010)

Keywords:
Odorant receptor; Heterologous functional expression; HEK293; Calcium imaging; Galpha; RTP; OR-S6

Materials & Methods:
HEK293 cells (human embryonal kidney 293 cells JCRB9068) were purchased from the Japanese Cancer Research Resource Bank (JCRB) Cell Bank through the Health Science Research Resource Bank (Osaka Japan). The HEK293 cells were cultured and allowed to proliferate following which they were frozen with freezing medium containing 90% heatinactivated fetal bovine serum (FBS) and 10% dimethyl sulfoxide in stock tubes at −80 ◦C and stored in liquid N2. ORS6 ORS83 (Malnic et al. 1999) ORcarc5 (MOR1302) ORcarn266 (MOR20237) or ORcarn270 (MOR1862) (Zhang and Firestein 2002; Hamana et al. 2003) was expressed in HEK293 cells (Krautwurst et al. 1998) along with RTP1 RTP2 (Saito et al. 2004) and Ga15 (Kajiya et al. 2001) or Ga15 olf. HEK293 cells were grown in culture dishes containing Dulbecco’s modified essential medium (DMEM) with 10% FBS without antibiotics at 37 ◦C under a humidified atmosphere containing 5% CO2 and were passaged every 2 or 3 days before the cells reached confluency. Confluent cells (60–80%) cultured in polydlysinecoated glassbottomed 35mm dishes (MatTek Co. Ashland MA USA) were transfected with 2.0mg RhoOR/ phCNV1 0.5mg RTP1/phCMV1 0.5mg RTP2/phCMV1 and 1.0mg Ga15 olf/pBKCMV (or Ga15/pBKCMV) by using 8ml lipofectamin 2000 (Invitrogen Carlsbad CA USA) according to manufacturer’s instructions.

Microscopic Technique
Epifluorescence microscopy

Cell Type(s)
HEK293 cells