새로운 Mammalian Expression vector EF1αPromoter Vector …

1 2 continued . Mammalian Expression Vector EF1 Promoter Vector . Human elongation factor-1 alpha (EF1 alpha) human (2) Lentiviral system . constitutive promoter , in vitro , in vivo . 1). , EF1 promoter . silencing Mouse ES cell line E14, D3 Clontech Lenti-X Lenviral system . 2). Takara Bio Clontech CMV promoter EF1 promoter AcGFP1. EF1 Promoter Vector plasmid . 5 FACS FL1 channel . AcGFP1 . EF1 promoter . (1) Mouse ES cell transient stable Expression cell line CMV promoter EF1 promoter . AcGFP1 ( 2). Wang et al ., 2008 CMV promoter EF1 . AcGFP1 pBApo-EF1 Neo pBApo-CMV Neo promoter silencing . (TaKaRa Code 3240) Xfect Stem Transfection Reagent (TaKaRa . Code 631320) mouse ES (E14TG2a) transient . 48 E14TG2a Flow cytometer AcGFP1 (transient Expression ).

2 % positive , positive . AcGFP1 MFI (Mean of Fluorescent Intensity) .. , 1:40 , G418 (TaKaRa Code 631307) 250 / .. 2 . Flow cytometer . AcGFP1 (stable Expression ).. Transient Expression EF1 promoter CMV promoter . (MFI ) . Stable Expression EF1 promoter (MFI . ), ES (E14TG2a) EF1 promoter . ( 1). Transient Expression Stable Expression 2. Expression of AcGFP1 driven by the EF-1 alpha promoter in stem cell lines is higher than Expression driven by the CMV promoter. The mouse embryonic stem cell lines E14 (Panel A) and D3 (Panel B) were transduced by Lenti-X lentivirus, expressing AcGFP1 either under the control of the CMV promoter or the Elongation factor alpha (EF-1 alpha) promoter. The Expression level of AcGFP1 in infected cells 5 days post infection was monitored by FACS analysis using the FL1 channel.

3 The Expression of AcGFP1 driven by the EF-1 alpha promoter in both stem cell lines was considerably higher compared to the CMV promoter. This is mainly due to a considerably lower rate of silencing of the EF-1 alpha promoter in stem cells com- 1. Mouse ES transient Expression stable Expression pared to the CMV promoter as published by Wang et al., 20083). 34 Life Science & Biotechnology 2 continued . EF1 Promoter Vector . (1) Takara Bio pBApo-EF1 DNA TaKaRa Code Takara Bio EF1 Promoter Mammalian cell pBApo-EF1 Neo DNA 20 3243. Expression Vector pBApo-EF1 Neo DNA (TaKaRa Code 3243) pBApo-EF1 Pur DNA 20 3244. pBApo-EF1 Pur DNA (TaKaRa Code 3244) .. pEF1 -IRES Vector 10 631970. Vector EF1 promoter Herpes Simple Virus Thymidine Kinase pEF1 -IRES-AcGFP1 Vector 10 631971.

4 PolyA signal . Cloning site ORF. pEF1 -IRES-DsRed-Express2 Vector 10 631980. Expression plasmid , . pEF1 -IRES-ZsGreen1 Vector 10 631976. pri-microRNA . pEF1 -AcGFP1-N1 Vector 10 631973.. Mammalian cell selection marker neomycin puromycin , Vector pEF1 -AcGFP1-C1 Vector 10 631974. ( 3). , Vector promoter ORF polyA signal pEF1 -DsRed-Monomer-N1 Vector 10 631978. cassette adenovirus Vector advenoviral pEF1 -DsRed-Monomer-C1 Vector 10 631977. delivery system . adenovirus pEF1 -DsRed-Express2 Vector 10 631979. Adenovirus Expression Vector Kit (Dual Version) Ver. 2 pEF1 -mCherry-N1 Vector 10 631969. (TaKaRa Code 6170) . pEF1 -mCherry-C1 Vector 10 631972. pEF1 -tdTomato Vector 10 631975. pEF1 -E2-Crimson Vector 10 631981.

5 PLVX-EF1 -IRES-Puro Vector 10 631988. pLVX-EF1 -IRES-mCherry Vector 10 631987. pLVX-EF1 -IRES-ZsGreen1 Vector 10 631982. pLVX-EF1 -AcGFP1-N1 Vector 10 631983. pLVX-EF1 -AcGFP1-C1 Vector 10 631984. pLVX-EF1 -mCherry-C1 Vector 10 631985. pLVX-EF1 -mCherry-N1 Vector 10 631986. pLVX-EF1 -DsRed-Monomer-C1 Vector 10 631989. 3. pBApo-EF1 Neo DNA, pBApo-EF1 Pur DNA pLVX-EF1 -DsRed-Monomer-N1 Vector 10 631990. pEF1 -Myc Vector 10 631991. Clontech EF1 Promoter Vector . pEF1 -HA Vector 10 631992. Clontech EF1 Promoter . pBApo-CMV Neo DNA 20 3240. , IRES (internal ribosome entry sequence) 2. pBApo-CMV Pur DNA 20 3241. , Myc, His tag . pBApo-CMV DNA 20 3242. Vector ( 4). Vector transfection . Adenovirus Expression Vector Kit (Dual plasmid Lentivirus delivery system 1 kit (5 ) 6170.)

6 Version) Ver. 2. lentiviral Vector . pAcGFP1-N1 Vector 20 632469. pAcGFP1-C1 Vector 20 632470. Xfect 100 /300 631317/631318. Xfect Stem 100 /300 631320/631321. Puromycin 25 /100 631305/631306. G418 1 g/5 g 631307/631308.. 1. Teschendorf, C., et al . (2002) Anticancer Res . 22(6A):3325-3330. 4. EF-1 alpha Expression vectors for many applications. Plasmid and lentiviral 2. Kim S, et al . (2007) Stem Cells Dev . 16(4):537-45. Vector choices are available carrying the EF-1 alpha promoter. IRES: internal ribo- 3. Wang, et al . (2008) Stem Cells Dev . 17:279 289. some entry sequence; FP1: fluorescent protein (AcGFP1, DsRed-Monomer, or mCherry); FP2: fluorescent protein (mCherry or ZsGreen1); MCS: multiple cloning *License Notice : [7], [8].

7 Site. September 2010 35.