Materials and Reagents

1. Mouse embryonic fibroblasts (prepared from day 14.5 embryos)
   
2. Vector pGL4.14 (Luc2 encoding vector) (Promega Corporation, catalog number: E6691 )
   
3. Primers (Invitrogen custom DNA primers)
   1.  5’-TAATACGACTCACTATAGGCCACCATGGAAGATGCCAAAAA-3’ (The T7 class III promoter sequence is underlined.)
      
   2.  5’-TACCACATTTGTAGAGGTTTTACTTGCTTT-3’
      
4. rTaq DNA polymerase (TOYOBO, catalog number: TAP-211 )
   
5. Agarose gel
   
6. Ethidium bromide
   
7. Illustra GFX PCR and Gel Band Purification Kit (GE, catalog number: 28-9034-71 )
   
8. MEGAScript In vitro transcription Kit (Life Technologies, Ambion^®, catalog number: AM1333 )
   Note: Nuclease-free Water and LiCl Precipitation Solution are included in the kit.
   
9. 80% ethanol
   
10. ScriptCap m7G capping system (EpiCentre, catalog number: SCCE0610 )
    
11. ScriptCap 2’-O-Methyltransferase Kit (EpiCentre, catalog number: SCMT0610 )
    
12. RNeasy Mini Kit (QIAGEN, catalog number: 74104 )
    
13. Opti-MEM I reduced serum medium (Life Technologies, Gibco^®, catalog number: 31985-070 )
    
14. D-PBS(-) (Nacalai Tesque, catalog number: 14249-95 )
    
15. Lipofectamine 2000 DNA Transfection Reagent (Life Technologies, catalog number: 11668-019 )
    
16. Dual-luciferase reporter assay system (Promega Corporation, catalog number: E1960 )
    
17. BCA protein assay reagent (Thermo Fisher Scientific, catalog number: 23227 )
    

Equipment

1. Cell scraper
   
2. GeneAmp PCR system 9700 (Applied Biosystems^®)
   
3. Centrifugal Concentrator CC105 (TOMY)
   
4. Lumat LB 9507 Luminometer (Berthold Technologies)
   
5. Model 680 Microplate Reader (Bio-Rad Laboratories)
   

Procedure