WO1993010245A1 - Nouveaux genes rapporteurs - Google Patents
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- WO1993010245A1 WO1993010245A1 PCT/EP1992/002582 EP9202582W WO9310245A1 WO 1993010245 A1 WO1993010245 A1 WO 1993010245A1 EP 9202582 W EP9202582 W EP 9202582W WO 9310245 A1 WO9310245 A1 WO 9310245A1
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- Prior art keywords
- gene
- protein
- reporter gene
- expression system
- expression
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- 108700008625 Reporter Genes Proteins 0.000 title claims abstract description 36
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 38
- 230000014509 gene expression Effects 0.000 claims abstract description 21
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 17
- 102000004190 Enzymes Human genes 0.000 claims abstract description 14
- 108090000790 Enzymes Proteins 0.000 claims abstract description 14
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 9
- 239000013612 plasmid Substances 0.000 claims description 14
- 239000013598 vector Substances 0.000 claims description 10
- 238000011144 upstream manufacturing Methods 0.000 claims description 8
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 6
- 101150018339 lacS gene Proteins 0.000 claims description 6
- 108010005774 beta-Galactosidase Proteins 0.000 claims description 4
- 102000005936 beta-Galactosidase Human genes 0.000 claims description 3
- 241000205091 Sulfolobus solfataricus Species 0.000 claims description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 abstract description 2
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 17
- 230000000694 effects Effects 0.000 description 14
- 238000013518 transcription Methods 0.000 description 8
- 230000035897 transcription Effects 0.000 description 8
- 108010035563 Chloramphenicol O-acetyltransferase Proteins 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- 108020004414 DNA Proteins 0.000 description 6
- 238000003556 assay Methods 0.000 description 6
- 108060001084 Luciferase Proteins 0.000 description 5
- 239000005089 Luciferase Substances 0.000 description 5
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 5
- 229960005091 chloramphenicol Drugs 0.000 description 5
- 230000002255 enzymatic effect Effects 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 230000002103 transcriptional effect Effects 0.000 description 5
- 241000588724 Escherichia coli Species 0.000 description 4
- ZSLZBFCDCINBPY-ZSJPKINUSA-N acetyl-CoA Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCSC(=O)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 ZSLZBFCDCINBPY-ZSJPKINUSA-N 0.000 description 4
- 108020004999 messenger RNA Proteins 0.000 description 4
- 238000003146 transient transfection Methods 0.000 description 4
- 108091026890 Coding region Proteins 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 230000000977 initiatory effect Effects 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 238000003153 stable transfection Methods 0.000 description 3
- 238000004809 thin layer chromatography Methods 0.000 description 3
- 230000001052 transient effect Effects 0.000 description 3
- 102000009572 RNA Polymerase II Human genes 0.000 description 2
- 108010009460 RNA Polymerase II Proteins 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 229940100228 acetyl coenzyme a Drugs 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 101150055766 cat gene Proteins 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 239000003623 enhancer Substances 0.000 description 2
- 210000002919 epithelial cell Anatomy 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 230000002452 interceptive effect Effects 0.000 description 2
- 210000004962 mammalian cell Anatomy 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- QTXZASLUYMRUAN-QLQASOTGSA-N Acetyl coenzyme A (Acetyl-CoA) Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCSC(=O)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1.O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCSC(=O)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 QTXZASLUYMRUAN-QLQASOTGSA-N 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- IGXWBGJHJZYPQS-SSDOTTSWSA-N D-Luciferin Chemical compound OC(=O)[C@H]1CSC(C=2SC3=CC=C(O)C=C3N=2)=N1 IGXWBGJHJZYPQS-SSDOTTSWSA-N 0.000 description 1
- CYCGRDQQIOGCKX-UHFFFAOYSA-N Dehydro-luciferin Natural products OC(=O)C1=CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 CYCGRDQQIOGCKX-UHFFFAOYSA-N 0.000 description 1
- QSJXEFYPDANLFS-UHFFFAOYSA-N Diacetyl Chemical group CC(=O)C(C)=O QSJXEFYPDANLFS-UHFFFAOYSA-N 0.000 description 1
- 108050001049 Extracellular proteins Proteins 0.000 description 1
- BJGNCJDXODQBOB-UHFFFAOYSA-N Fivefly Luciferin Natural products OC(=O)C1CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 BJGNCJDXODQBOB-UHFFFAOYSA-N 0.000 description 1
- 108700028146 Genetic Enhancer Elements Proteins 0.000 description 1
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 241000254158 Lampyridae Species 0.000 description 1
- DDWFXDSYGUXRAY-UHFFFAOYSA-N Luciferin Natural products CCc1c(C)c(CC2NC(=O)C(=C2C=C)C)[nH]c1Cc3[nH]c4C(=C5/NC(CC(=O)O)C(C)C5CC(=O)O)CC(=O)c4c3C DDWFXDSYGUXRAY-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- 241001045988 Neogene Species 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 241000254064 Photinus pyralis Species 0.000 description 1
- 108091034057 RNA (poly(A)) Proteins 0.000 description 1
- 108700026226 TATA Box Proteins 0.000 description 1
- 108700009124 Transcription Initiation Site Proteins 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 1
- 230000021736 acetylation Effects 0.000 description 1
- 238000006640 acetylation reaction Methods 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- WQZGKKKJIJFFOK-FPRJBGLDSA-N beta-D-galactose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-FPRJBGLDSA-N 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 210000004671 cell-free system Anatomy 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000000975 co-precipitation Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 239000003398 denaturant Substances 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000000326 densiometry Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- LIYGYAHYXQDGEP-UHFFFAOYSA-N firefly oxyluciferin Natural products Oc1csc(n1)-c1nc2ccc(O)cc2s1 LIYGYAHYXQDGEP-UHFFFAOYSA-N 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000001744 histochemical effect Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000005567 liquid scintillation counting Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 101150091879 neo gene Proteins 0.000 description 1
- 125000001209 o-nitrophenyl group Chemical group [H]C1=C([H])C(*)=C(C([H])=C1[H])[N+]([O-])=O 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- JJVOROULKOMTKG-UHFFFAOYSA-N oxidized Photinus luciferin Chemical compound S1C2=CC(O)=CC=C2N=C1C1=NC(=O)CS1 JJVOROULKOMTKG-UHFFFAOYSA-N 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 230000008488 polyadenylation Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000005026 transcription initiation Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000011830 transgenic mouse model Methods 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01023—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/67—General methods for enhancing the expression
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2468—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on beta-galactose-glycoside bonds, e.g. carrageenases (3.2.1.83; 3.2.1.157); beta-agarase (3.2.1.81)
- C12N9/2471—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
Definitions
- RNA polymerase II catalyzes the synthesis of mRNA from the structural DNA genes through the formation of a complex with specific target DNA sequences (promoters) and protein factors which are responsible for the correct initiation of the transcription. Promoters for RNA polymerase II contain a variety of short cis-acting DNA sequences which are specifically recognised by transacting protein factors. These modular DNA sequences are located mainly upstream of the TATA box (when present) but can be placed in both orientations and at very variable distances from the transcription start site.
- the proximal region to this site is responsible for correct initiation of transcription, while the regions further upstream determine the transcriptional efficiency of the overall promoter.
- Some of these promoter sequences are contained in several genes and are recognised by widely present protein factors.
- Other promoter sequences are characteristic of specific groups of genes and interact with specific transcription factors: they are called "responsive elements" (RE) .
- RE responsive elements
- Reporter genes code for proteins which possess a unique enzymatic activity or are otherwise easily distinguishable from the mixture of intra- and extra- cellular proteins produced by the expression system under study.
- the basic strategy for analysing the efficiency of gene transcription using reporter genes is as follows. To test the efficacy of the promoter elements of the expression system, the DNA under test is ligated upstream of the coding region of the reporter gene so as to produce a chi eric gene in which the putative regulatory elements control the expression of the reporter gene.
- functional enhancer elements which by definition can increase transcription when placed upstream or downstream and in either orientation relative to the promoter, the test DNA can be ligated upstream or downstream of a reporter gene which already is associated with a promoter.
- the fused genes so obtained are subsequently introduced, by any of a variety of techniques, into cultured cells or into animals, e.g. to produce transgenic mice.
- the transcriptional capability of the DNA under test is then estimated quantitatively from the in vitro activity of the reporter gene product in the culture medium or in cellular or tissue-derived extracts, or is determined qualitatively or by histochemical techniques of intact cells.
- the basic technique may be varied to take account of the particular system being studied.
- the activity or quantity of the reporter gene product is an indirect measurement of the transcriptional properties of the DNA under test, generally the reporter gene activity is directly proportional to the transcriptional activity.
- the vectors containing reporter genes used up till now have certain properties in common. They often have a backbone derived from pBR322 or pUC, which allow for propagation and selection in Escherichia coli.
- such vectors generally possess a eucaryotic poly(A) addition signal and an intron, either intrinsic to the reporter gene or derived from a heterologous source. The presence of an intron is necessary for efficient production of some mature cytoplasmic mRNAs.
- Unique cloning sites, located upstream or downstream of the reporter gene, are designed for easy insertion of promoter and RE sequences to be tested.
- Chloramphenicol Acetyltransferase One widely used reporter gene codes for chloramphenicol acetyltransferase (CAT) in E. coli(l) .
- the CAT gene is not endogenous to mammalian cells and the enzyme which it produces can be easily assayed and its activity detected at low levels.
- CAT catalyses the transfer of acetyl groups from acetyl coenzyme A (acetyl-CoA) to chloramphenicol. Mono- and diacetyl forms of chloramphenicol can be produced.
- acetylated and unacetylated forms of chloramphenicol possess different solubilities in organic solvents, they can be separated by silica gel thin-layer chromatography (TLC) or by differential extraction in organic solvents.
- the acetylated forms can be radiolabelled by incubating ( 14 C)chloramphenicol with acetyl-CoA, or, less expensively, incubating ordinary chloramphenicol with ( 3 H)acetyl-CoA in the test reaction.
- the amount of acetylation can be measured by densitometry or by liquid scintillation counting of the appropriate region of the silica gel after TLC or the organic phase after differential extraction.
- (b) Luciferase The DNA sequence coding firefly (Photinus pyralis) luciferase can be used in a very versatile reporter gene system (2,3). Luciferase catalyses the reaction:
- both the maximum light intensity and the total light output over a period of time are proportional to the concentration of luciferase in the reaction system.
- the light emission can be measured by a luminometer or by liquid scintillation.
- Beta-Galactosidase S-galactosidase from E. coli is a tetramer with a subunit size of 1023 amino acids. It catalyses the hydrolysis of various ⁇ -galactosides, including lactose. Measurement of /9-galactosidase activity provides a useful internal control for measuring the variability in reporter enzyme activity caused by differences in transfection efficiency or cell extract preparation(4) . A simple photometric assay method may be used to measure hydrolysis of the substrate ONPG (o- nitrophenyl SD-galactopyranoside) by /9-galactosidase in a cell-free system. The recent discovery of substrates for 9-galactosidase which generate fluorescent products has significantly increased the sensitivity of this assay.
- ONPG o- nitrophenyl SD-galactopyranoside
- mRNA from the reporter gene and consequently enzymatic activity can be generated by read-through transcription or by spurious initiation of the transcription from within the plasmid-associated procaryotic sequences. It has been demonstrated that many plasmid constructions containing CAT as reporter gene may drive transcription initiation at several unspecific sites, leading to spuriously increased CAT activity. In addition, the assay method is quite complicated and not easy to reproduce. Use of radio labelled compounds requires special laboratory licences.
- a recombinant protein or peptide expression system in accordance with the present invention comprises, as a reporter gene, a gene coding for an enzyme or other protein which is stable under conditions, e.g. of temperature, in which at least some, and preferably all, of the other protein or peptide products of the said system are denatured or otherwise inactivated.
- the expression system includes, in addition to the reporter gene, a promoter and/or other DNA sequences which do not encode a protein or peptide product but assist in the expression of the reporter gene.
- genes coding for highly thermostable enzymes represents a novel and advantageous strategy for the study of the efficiency of expression of eucaryotic cis-acting genomic sequences.
- Using reporter genes which give rise to products which are intrinsically stable to temperature and protein denaturants allows the quick and efficient removal, by suitable selective denaturation, of any interfering proteins which are endogenously expressed by the host cells.
- the new systems may be used in cell systems which endogenously express high levels of an interfering enzyme
- the detection system can be made to have high specificity which can be as high as that of a direct measure of the mRNA corresponding to the reporter gene.
- thermostable enzymes are already known, especially those present in extremophile achae-bacteria.
- the thermostable enzymes have been well characterized (5,6,7,8) and the corresponding genes coding for them are available (9) .
- the gene (lacS) encoding a ⁇ - galactosidase (SsB-Gal) from the archeabacterium Sulfolobus solfataricus is preferably used in the present invention.
- This microorganism, strain MT4 is available from, for example, ATCC and DSM
- the enzyme produced by this gene has been purified to homogeneity and extensively characterized (8) .
- a gene library of the microorganism has been constructed in lambda gtll and the /9-galactosidase gene isolated, cloned (9) and expressed in E. coli (10) .
- the potency of lacS as a reporter gene in eukaryotic systems has been demonstrated in cultured mammalian cells.
- four different eukaryotic expression vectors, suitable for transient or stable transfection have been constructed. All four vectors contain strong enhancer/promoter sequences which allow high expression of the cloned gene.
- Figures 1 and 2 are plasmid maps of the four vectors which have been made. The first two plasmids suitable for transient transfection (see Figure 1) were designed as follows.
- This plasmid was constructed by substituting the Hind III/Pvu II fragment of the CAT gene in pSV 2 -CAT(l) using a Hind III fragment containing all the coding sequence of the lacS gene.
- pRSV-lacS
- the Bam HI region (containing the lacS gene and the SV40 polyadenylation site and intron) from pSV 2 -lacS was inserted in the Hind III/Bam HI digested pRSV-neo(l) , replacing the neo coding sequence by the sequence containing the lacS gene.
- Two more plasmids were constructed for the selection of stable transfectants. These are shown in Figure 2. Both the plasmids possess the neo gene under the control of the SV40 enhancer/promoter system.
- pRC/RSV-lacS and pRC/CMV-lacS were constructed by inserting the Hind III fragment of LacS at the Hind III site of the polylinkers of, respectively, pRC/RSV and pRC/CMV vectors obtained from INVITROGEN.
- Simian CV1 and mouse fibroblast NIH3T3 cell lines were used as host systems. They were transfected by the calcium/phosphate coprecipitation technique. For transient invention, cells were harvested 48 hours after DNA addition and cell lysates were prepared and assayed for enzymatic activity. For stable transfection CV1 cells transfected with pRC/CMV-lacS were selected by growth in the presence of selection marker G418. To assay the expressed Ss 0-gal activity, cell extracts were preincubated at 70*C for ten minutes in the presence of 0.1% SDS to inactivate any endogenous enzymatic activity of the transfected cells. The assay was then performed by the method of Maniatis et al (11) but at 75°C rather than 37°C.
- thermostable enzymes in the two cell lines are shown in Figure 3.
- Non- transfected NIH3T3 and CV1 cells were used as negative controls giving background activity values.
- Ss . ⁇ -gal activity was reported in relation to cell number and normalised with respect to CAT activity determined in cells which were cotransfected with the pSV 2 CAT plasmid.
- NIH3T3 cells were transfected with 2 ⁇ g of pSV -CAT or alternatively with 2 or 20 ⁇ g of pRSV-lacS or pRC/RSV-lacS.
- CVl cells were cotransfected with the same amount of the reference plasmid or with 2 or 20 ⁇ g of pSV 2 -lacS or pRC/CMV.
- the expression system of the present invention is conveniently provided in the form of a plasmid or other vector comprising the said system, preferably with a polylinker sequence upstream of the reporter gene to facilitate cloning of a desired promoter region to be tested.
- plasmids may be used in transient or stable transfection.
- plasmids or other vectors may be provided having an expression system in which the same reporter gene is under the control of a promoter sequence of known potency.
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- Engineering & Computer Science (AREA)
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- General Engineering & Computer Science (AREA)
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- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
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Abstract
Système d'expression de protéine ou de peptide de recombinaison comprenant, sous forme de gène rapporteur, un gène codant pour une enzyme ou autre protéine qui est stable dans des conditions, par exemple, de température, dans lequel au moins quelques-uns et, de préférence, tous les autres produits protéiniques ou peptidiques du système sont neutralisés.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB9123987.1 | 1991-11-12 | ||
GB919123987A GB9123987D0 (en) | 1991-11-12 | 1991-11-12 | New reporter genes |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1993010245A1 true WO1993010245A1 (fr) | 1993-05-27 |
Family
ID=10704474
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/EP1992/002582 WO1993010245A1 (fr) | 1991-11-12 | 1992-11-10 | Nouveaux genes rapporteurs |
Country Status (2)
Country | Link |
---|---|
GB (1) | GB9123987D0 (fr) |
WO (1) | WO1993010245A1 (fr) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1998036097A1 (fr) * | 1997-02-14 | 1998-08-20 | Ventana Genetics, Inc. | Procedes pour identifier, caracteriser et faire evoluer des elements regulateurs a effet cis specifique de type cellulaire |
WO1999002737A1 (fr) * | 1997-07-14 | 1999-01-21 | Valentis, Inc. | Procede d'identification de regions de synthese regulatrices de transcription specifiques de cellules ou de tissus |
US6566057B1 (en) | 1997-02-14 | 2003-05-20 | Deltagen Proteomics, Inc. | Methods and compositions for peptide libraries displayed on light-emitting scaffolds |
US6579675B2 (en) | 1997-02-14 | 2003-06-17 | Deltagen Proteomics, Inc. | Methods for identifying nucleic acid sequences encoding agents that effect cellular phenotypes |
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1991
- 1991-11-12 GB GB919123987A patent/GB9123987D0/en active Pending
-
1992
- 1992-11-10 WO PCT/EP1992/002582 patent/WO1993010245A1/fr active Application Filing
Non-Patent Citations (7)
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CHEMICAL ABSTRACTS, vol. 114, no. 21, 27 May 1991, Columbus, Ohio, US; abstract no. 205470x, M. MORACCI ET AL. 'Cloning and expression as a tool to easily purify a new highly thermostable archebacterial beta-galactosidase' page 666 ;column R ; * |
GENE vol. 106, no. 1, 30 September 1991, ELSEVIER SCI. PUBL., AMSTERDAM, NL; pages 13 - 19 G. BURCHHARDT AND H. BAHL 'Cloning and analysis of the beta-galactosidase-encoding gene from Clostridium thermosulfurogenes EM1' * |
GENE vol. 94, no. 2, 15 October 1990, ELSEVIER SCI. PUBL., AMSTERDAM, NL; pages 89 - 94 M.V. CUBELLIS ET AL. 'Isolation and sequencing of a new beta-galactosidase-encoding archebacterial gene' * |
J. BACTERIOL. vol. 174, no. 3, February 1992, AM. SOC. MICROBIOL.,BALTIMORE, US; pages 873 - 882 M. MORACCI ET AL. 'Expression of the thermostable beta-galactosidase gene from the archaebacterium Sulfolobus solfataricus in Saccharomyces cerevisae and characterization of a new inducible promoter for heterologous expression' * |
MOLECULAR CLONING, A LABORATORY MANUAL 1989, CSH LABORATORY PRESS,LONG ISLAND,US; pages 16.56 - 16.59 SAMBROOK, FRITSCH, MANIATIS 'Srategies for studying gene regulation' cited in the application * |
NATO ASI SERIES A: LIFE SCIENCES vol. 183, 1989, SPRINGER VERLAG, BERLIN, BRD; pages 325 - 331 M.ROSSI ET AL. 'Cloning, sequencing and expression of a new beta-galactosidase from the extreme thermophilic sulfolobus solfataricus' cited in the application * |
NUCLEIC ACID RESEARCH vol. 17, no. 19, 11 October 1989, EYNSHAM, OXFORD, GB; page 7980 S. LITTLE ET AL. 'Nucleotide sequence of a thermostable beta-galactosidase from Sulfolobus solfatarius' the whole document * |
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US6566057B1 (en) | 1997-02-14 | 2003-05-20 | Deltagen Proteomics, Inc. | Methods and compositions for peptide libraries displayed on light-emitting scaffolds |
US6579675B2 (en) | 1997-02-14 | 2003-06-17 | Deltagen Proteomics, Inc. | Methods for identifying nucleic acid sequences encoding agents that effect cellular phenotypes |
US6623922B1 (en) | 1997-02-14 | 2003-09-23 | Deltagen Proteomics | Methods for identifying, characterizing, and evolving cell-type specific CIS regulatory elements |
WO1999002737A1 (fr) * | 1997-07-14 | 1999-01-21 | Valentis, Inc. | Procede d'identification de regions de synthese regulatrices de transcription specifiques de cellules ou de tissus |
US6410228B1 (en) | 1997-07-14 | 2002-06-25 | Baylor College Of Medicine | Method for the identification of synthetic cell- or tissue- specific transcriptional regulatory regions |
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