遺伝子発現に重要なベクターの要素 - lonzabio.jp

1 1 Amaxa .. CMV .. CMV . 1 . CAT .. 100% CMV . 10 .. BHK-21 . 1 . SV40 .. 10 .. mRNA .. A. 1600. Relative luciferase-expression % of [pGL3-CMV at 24 h]. 1400. 1200. 1000. 800. 600. 400. 200. 0. Control Program pGL3- pGL3-CMV pmax pSG5 pFP pIRES- pcDNA4 pCMVbeta pCMV-TNT. only control Cloning MCS B HisMax B. 2000. Relative luciferase-expression % of [pGL3-CMV at 24 h]. 1500. 1000. 500. 0. Control Program pGL3- pGL3-CMV pmax pSG5 pFP pIRES- pcDNA4 pCMVbeta pCMV-TNT. only control Cloning MCS B HisMax 4 hours 24 hours 48 hours 1. Luciferase expression levels depend on vector backbones.

2 We looked at luciferase expression at 4, 24 and 48 hours in THP-1 and HUVEC cells. The amount of DNA was held at equimolar amounts based on plasmid size. For THP-1, the DNA amount ranged from g per reaction (A). For HUVECs, we used g of plasmid (B). 03-6264-0660 . 03-6264-0660 03-6264-0660 03-6264-0620 LBS-W0001-030.. 1 . SV40 EF1a RSV CMV . 293 5% 74% 100% 4, 6. BHK-21 200% 200% 100% 4. C6 44% 100% 5. CHO-K1 16% 11% 100% 2, 4, 5. Cos-7 7% 15% 7% 100% 1, 2, 4, 5, 8. HeLa 43% 73% 29% 100% 2, 3, 4, 5, 6, 8. N2A (Neuro-2A) 50% 100% 5.. NIH-3T3 67% 143% 107% 100% 2, 3, 7, 8. IRES . IRES 2 IRES.

3 GFP. GF P 2 . IRES . mRNA 2 . mRNA IRES MCS . mRNA A IRES . 2 B 2 GFP . pIRES Clontech . IRES 2B GFP IRES GFP . GFP . GFP GFP . A. pmaxGFP Vector: CMV* pmaxGFP Vector Construct A: CMV* pmaxGFP Vector IRES MCS B empty Construct B: CMV* MCS B empty IRES pmaxGFP Vector B. Expression in HL-60. Expression in Huvec HL-60 HUVEC . Mean fluorescence (% of pmaxGFP vector). 120 120. Mean fluorescence (% of pmaxGFP vector). 100 100. 80 80. 60 60. 40 40. 20 20. 0 0. pmaxGFP Vector Construct A Construct B pmaxGFP Vector Construct A Construct B. 2. Reporter gene expression is dependent on the position in an IRES expression vector.

4 HL-60 and HUVEC cells were transfected by Nucleofection with either pmaxGFP Vector or pIRES variants containing maxGFP Reporter Protein cloned in either the Multiple Cloning Site (MCS) upstream (construct A) or downstream of the IRES sequence (construct B; 2A). Figure (2B) shows reduced GFP expression using IRES plasmids especially if GFP is located downstream of the IRES sequence. 03-6264-0660 . 03-6264-0660 03-6264-0660 03-6264-0620 LBS-W0001-031.. 4 7 kb . Nucleofection . IRES 20 kb . 2 . mRNA .. Nucleofection .. BAC . IRES 9 10.. LTR .. LTR .. LTR . Nucleofection 6 16 .. Nucleofection 24.

5 Nucleofection .. CMV pmaxCloning Vector . EF1a SV-40 mRNA . Nucleofection . gal .. Kinetics of Luciferase Expression in HUVEC. HUVEC . (Protein/Well, 24 hours value = 100%). HUVEC. Kinetics of -gal -gal Expression . in HUVEC. (Protein/Well, 24 hours value = 100%). / 24 =100% / 24 =100% . 300 300. Relative expression per cell (% of 24 hours value). Relative expression per cell (% of 24 hours value). 250 250. 200 200. 150 150. 100 100. 6 16 hours 10 48 hours 50 50. 0 0. 0 10 20 30 40 50 0 10 20 30 40 50. Time post-transfection (hours) Time post-transfection (hours). 3. Expression kinetics are reporter gene dependent.

6 HUVEC cells were transfected by Nucleofection with either a luciferase or a -gal expression vector. While luciferase expression shows a maximum 6 16 hours post-transfection, -gal expression is sustained for several days after transfection. 03-6264-0660 . 03-6264-0660 03-6264-0660 03-6264-0620 LBS-W0001-032.. 3 vs.. gal HUVEC .. 16 . gal 10 1 . 24 GFP 2 . 1 . 2 .. Nucleofection 6 16 .. RNA . gal GFP Nucleofection 10 48 .. GFP . ATG .. 1 . ATG .. 1. Cheng et al., (1995) Int J Radiat Biol 67(3): 261-267. 2. Foecking et al., (1986) Gene 45(1): 101-105. 3. Davis et , (1988) Biotechnol Appl Biochem 10(1): 6-12.

7 4. Liu et al., (1997) Anal Biochem 246(1): 150-152. 5. Wenger et al., (1994) Anal Biochem 221(2): 416-418. 6. Kronman et al., (1992) Gene 121(2): 295-304. 7. Thompson et al., (1993) In Vitro Cell Dev Biol 29A(2): 165-170. 8. Thompson et al., (1990)Gene 96(2): 257-262. 9. Marshall et al., (2005) J Biol Chem 280(39): 33357-33367. 10. Wang et al., (2006) J Virol 80(12): 6003-6012. 03-6264-0660 . 03-6264-0660 03-6264-0660 03-6264-0620 LBS-W0001-033.