Mammalian gene expression is improved by use of a longer SV₄₀early polyadenylation cassette

Abstract

The expression efficiency of both cassettes was tested in two pUC-based expression vectors, that are otherwise comparable. In both constructs the respective polyadenylation sequences are fused to the coding sequences of the chloramphenicol acetyltransferase (CAT) gene. The expression of CAT activity was assayed when driven by a weak or by a strong promoter. As weak promoters the first 161 (PstI) or 525 bp (DraI) (4) nucleotides upstream of the transcription initiation site of the rat carbamoylphosphate synthase (CPS) gene were used, while the Rous sarcoma virus long terminal repeat (RSV-LTR) was used as a strong promoter. After CsCl purification, 50 Ag of these constructs was transfected into 1 x 107 FTO2B rat hepatoma cells as described (5). As an internal control 1 /tg pRSVluc was included in each transfection. Two days after electrotransfection, the cells were harvested in ice-cold PBS supplemented with 2 mM EDTA pH 8.0, and lysed in 0.1 % Triton X-100 in 100 mM potassium phosphate buffer pH 7.6. In each lysate the protein concentration (BCA protein reagent kit, Pierce), luciferase (6) and chloramphenicol acetyl transferase activity (7) was determined.Both vectors expressed no detectable CAT activity, when promoter sequences were absent. The activities of the weak promoters when expressed from the vector containing the shorter polyadenylation sequence are below the limit of detection of the assay, whereas when expressed in combination with the longer polyadenylation sequence a low but reproducible activity was detected (data not shown). When expression of the CPS promoter constructs was hormonally stimulated (10-7 M dexamethasone, 10-4 M dibutyryl cAMP and 10-3 M 3-isobutyl-1-methylxanthine), CAT expression became measurable even with the shorter polyadenylation sequence, but calculated activities were more than 100-fold lower than those obtained with the longer sequences for polyadenylation (Table 1). When CAT activity was driven by the strong viral RSV-LTR promoter, a 50-fold enhancement of gene expression was found with the longer polyadenylation sequence compared to the shorter (Table 1). A quantitatively comparable enhancement of expression of these constructs was observed in rat 5123 hepatoma cells, in rat Rat-I fibroblasts and in mouse NIH 3T3 fibroblasts. The shorter polyadenylation sequence in the late orientation has been shown to be more efficient than in the early orientation (10). If the shorter sequences for polyadenylation in the early orientation is extended up to the EcoRI site, gene expression proves to be at least equally if not more efficient than the short one in the late orientation.A convenient cloning vector should contain only the essential sequences necessary for high expression. The plasmid containing the RSV-LTR promoter upstream of the CAT gene and the longer polyadenylation sequence downstream of the CAT gene was used to identify the shortest sequence having the full capacity. The supercoiled plasmid was linearized with restriction enzymes BamHI, ApaI, PstI and NdeI to create seq...

A universal vector concept for a direct genotyping of transgenic organisms and a systematic creation of homozygous lines

“…In #3 and #4 of pAVOIAF{#1–#2–#3–#4}, mOrange- and mCherry-based eye-specific transformation markers (mO and mC, respectively) were inserted (in reverse orientation) that consist of (i) the artificial 3×P3 promoter ( Berghammer et al, 1999 ), (ii) the codon-optimized open-reading frames for the respective fluorescent protein, that is, mOrange2 ( Shaner et al, 2008 ) or mCherry ( Shaner et al, 2004 ) and (iii) the SV40 poly(A) ( van den Hoff et al, 1993 ). Both markers are flanked by incompatible Lox sites (the spacer is underlined, deviations are marked bold): upstream by a LoxP (5’-ATAACTTCGTATA G C ATAC A T TATACGAAGTTAT-3’) and downstream by a LoxN (5’-ATAACTTCGTATA G T ATAC C T TATACGAAGTTAT-3’) site.…”

“…In #2 of pAGOC, a modular fluorescent protein expression cassette was inserted, which consists of (i) a two-slot cloning site composed of a promoter (#P) and an open-reading frame (#O) slot, (ii) a 9 bp Ala-Ala-Ala linker, (iii) the codon-optimized mEmerald open-reading frame ( Tsien, 1998 ) and (iv) an elongated variant of the SV40 poly(A) ( van den Hoff et al, 1993 ). The #P slot can be accessed by the AscI/FseI site pair, or alternatively scarlessly by the double BtgZI site pair.…”

Next generation marker-based vector concepts for rapid and unambiguous identification of single and double homozygous transgenic organisms

“…S1A ), whose creation and further usage is described below. The vector contains two fluorescence-based and thus phenotypically distinguishable markers that consist of (i) the artificial 3×P3 promoter ( Berghammer et al, 1999 ), (ii) the open-reading frame for either mCerulean2 ( Markwardt et al, 2011 ) or mVenus ( Nagai et al, 2002 ), and the (iii) SV40 poly(A) ( van den Hoff et al, 1993 ). Both markers are flanked upstream by a LoxP ( Hamilton and Abremski, 1984 ) and downstream by a LoxN ( Livet et al, 2007 ) site, which results in interweaved, but incompatible Lox site pairs ( Fig.…”

“…S1B ). Further, it contains a modular fluorescent protein expression cassette, which consists of (i) a two-slot cloning site composed of a promoter (#P) slot and an open-reading frame (#O) slot, (ii) a 9 bp Ala-Ala-Ala linker, (iii) the codon-optimized mRuby2 ( Lam et al, 2012 ) open-reading frame, and (iv) an elongated variant of the SV40 poly(A) ( van den Hoff et al, 1993 ). The #P slot can be accessed by either the AscI / FseI site pair or scarlessly by the head-to-head-oriented double BtgZI site pair.…”