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WO1998016634A1 - Expression transgene - Google Patents

Expression transgene Download PDF

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Publication number
WO1998016634A1
WO1998016634A1 PCT/GB1997/002830 GB9702830W WO9816634A1 WO 1998016634 A1 WO1998016634 A1 WO 1998016634A1 GB 9702830 W GB9702830 W GB 9702830W WO 9816634 A1 WO9816634 A1 WO 9816634A1
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Prior art keywords
transgene
genetic construct
flanking
expression
transgenic
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PCT/GB1997/002830
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English (en)
Inventor
Anthony John Clark
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Biotechnology And Biological Sciences Research Council
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Priority to AU47109/97A priority Critical patent/AU4710997A/en
Publication of WO1998016634A1 publication Critical patent/WO1998016634A1/fr

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    • C—CHEMISTRY; METALLURGY
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    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4717—Plasma globulins, lactoglobulin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8121—Serpins
    • C07K14/8125—Alpha-1-antitrypsin
    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/103—Ovine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/01—Animal expressing industrially exogenous proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/80—Vector systems having a special element relevant for transcription from vertebrates
    • C12N2830/85—Vector systems having a special element relevant for transcription from vertebrates mammalian

Definitions

  • the invention relates to a genetic construct which causes improved expression of a protein and/or RNA coding sequence when the construct is introduced into a host organism, vectors which include the construct, transgenic organisms which provide improved expressed of such a coding sequence and a method for the production of such a transgenic organism.
  • Transgenic organisms are today common tools in areas of technology such as medicine, agriculture and food production.
  • One specific use, of considerable potential is of transgenic organisms as bioreactors for protein production.
  • Of particular benefit is the use of transgenic livestock as bioreactors for the production of human proteins .
  • transgenic organisms as bioreactors requires the ability to repeatedly provide transgenic organisms with high levels of transgene expression. To date, this has not been achieved. Observations in transgenic organisms are that the same transgene construct varies widely in its level of expression in different transgenic lines.
  • One theory for this course of variation is that the chromosomal sequence at the site of integration affects expression. This is referred to as the "position effect" and is discussed in WO 92/11358 and in Al-Shawi et al (Mol . Cell . Biol . 10, 1192-98 (1990)).
  • WO 92/11358 teaches that position effects could be overcome by integrating a poorly expressed transgene in the region of an efficiently expressed transgene. This enabled the poorly expressed coding sequence to overcome the negative position effects, and consequently, to exhibit enhanced expression. However, in order to achieve enhanced expression of a poorly expressed transgene in that case, cointegration of coding sequences was required.
  • transgenes are very highly expressed irrelevant of their site of integration in the host chromosome. Such transgenes are considered to "dominate” just any site into which they integrate and so overcome any effects mediated by the endogenous DNA. Unfortunately this ability to "dominate” and thus result in high expression is limited to a small number of transgenes .
  • the ability to produce high levels of expression of a selected transgene in lines of transgenic animals would be of enormous importance. Of additional importance would be a method for the production of transgenic lines which have some degree of commensurate and predictably enhanced transgene expression level.
  • the present invention contributes towards the achievement of such goals.
  • the present invention is based on the premise that in most cases endogenous sequences at the site of integration of a transgene may be directly responsible for the result of the observed "position effects". This follows the proposition that the site of integration of transgenes is random and that sequences in the host genome possess an inherent ability for expression of such integrated DNA (i.e. some sites in the genome are more permissive for expression than others) .
  • transgene would have integrated into a permissive site.
  • transgenes can be used as probes for the identification of such permissive and non-permissive sites in a host which has been transformed with that transgene.
  • a genetic construct comprising sufficient regulatory sequence operatively linked to a protein and/or RNA coding sequence to direct its expression and sufficient flanking DNA from a highly expressed transgenic loci to cause improved expression of the protein and/or RNA coding sequence when the construct is introduced into a host organism.
  • transgene is intended to include any nucleic acid sequence, including cDNA, which encodes all or part of any polypeptide (coding sequence) that is operatively linked to sufficient regulatory DNA sequences to direct its expression.
  • the nucleic acid may be derived from any source, including the genome of the host animal. Both of the terms “transgene” and “coding sequence” encompass nucleic acid sequences of foreign and endogenous derivation.
  • flanking DNA is intended to include all or any part of a sequence corresponding to the flanking sequences of any highly expressed transgenic loci which result in improved/enhanced expression of the transgene.
  • Corresponding sequences include identical sequences or variations thereof including, in particular, the replacement of a purine base by another purine base and the replacement of a pyrimidine base by another pyrimidine base which also result in improved/enhanced expression of the transgene.
  • the flanking DNA may comprise either all or part of the 5' flanking sequence, all or a part of the 3' flanking sequence or all or a part of both the 5' and 3' flanking sequences from a highly expressed transgenic loci .
  • Flanking sequences from transgenic loci can be isolated by conventional cloning techniques and vectors all of which are well known to those skilled in the art, including yeast artificial chromosomes (e.g. up to 1 megabase) , cosmid vectors or bacteriophages .
  • the flanking DNA is all or part of both of the cloned 5' and 3' flanking sequences. Incorporation of such flanking DNA results in the improved expression of the transgene.
  • the flanking sequence (s) are preferably located either side of the transgene, (ie the 5' flanking sequence is on the 5' side of the coding sequence and the 3' flanking sequence is on the 3' side of the coding sequence) .
  • flanking sequences from a highly expressed transgenic loci are either or both of the flanking sequence from the secondary transgene TAB (including TAB 3' and TAB 5') contained in the cell line deposited under the Accession No. NCIMB 40806.
  • Optimum expression levels of such a construct are obtained when both the 5 ' and 3 ' flanking sequences are present and preferably where the 5' flanking sequence is located on the 5' side of the transgene and the 3 ' flanking sequence is located on the 3' side of the transgene.
  • the construct comprises the murine derived flanking DNA of the secondary transgene TAB contained in the cell line deposited under the Accession No.
  • the coding sequence of the construct is preferably mammalian derived, most preferably human although it may have its derivation in one or more of any organism including humans, or non-human mammals, for example sheep, pigs and mice.
  • suitable coding sequences according to the invention include human derived genes, for example the human gene(s) which encode alpha 1-antitrypsin, milk proteins and blood factors such as Factors VIII and IX.
  • the host organism may be eukaryotic, including animal or plant.
  • the choice of host preferably reflects the derivation of the regulatory sequence of the transgene, i.e. a transgene containing regulatory sequences derived from a mammal is preferably expressed in a mammalian host, suitably a non-human mammalian host.
  • Suitable regulatory sequences operatively linked to the protein and/or RNA coding sequence (s) to direct its expression are well known in the art and can be selected according to requirement .
  • An example of a suitable regulatory DNA sequences is the beta-lactoglobulin promoter. This particular promoter drives expression of the protein encoded by the coding sequence in the mammary gland of transgenic mammals, preferably non-human transgenic mammals.
  • the selected coding sequence is targeted for expression to the mammary glands then it is essential that the host animal is a mammal.
  • Suitable laboratory mammals for experimental ease of manipulation include mice and rats. Larger yields may be obtained from domestic animals such as cows, pigs, sheep and other mammals. Such domestic farm animals and 'intermediate' animals such as rabbits are most suitable as bioreactors for protein production.
  • a vector which comprises a genetic construct according to any part of the first aspect of the invention.
  • Suitable vectors are commonplace in the art and include the well known vectors Lambda DASH II
  • an eukaryotic cell comprising a genetic construct according to any part of the first aspect of the invention.
  • the genetic construct is part of a mammalian cell, suitably a non-human mammalian cell, more preferably a murine or ovine cell, most preferably a mammary gland cell or a liver cell.
  • Methods for the introduction of genetic constructs into cells are well known in the art and include pronuclear injection and electroporation .
  • a transgenic organism comprising a genetic construct according to the first aspect of the invention integrated into its genome, preferably a non-human transgenic organism.
  • a transgenic organism can carry the integrated genetic material stably and which, as a result of the flanking sequence (s) associated with the transgene is capable of expressing at improved or enhanced levels, the integrated transgene.
  • the transgenic organism is preferably a sheep or a mouse.
  • a method of providing improved expression of a transgene from a transgenic host organism preferably a non-human transgenic organism.
  • the method comprises introducing into the host organism a genetic construct according to any part of the first aspect of the invention.
  • Techniques for introducing genetic constructs into organisms are well known in the art and include pronuclear injection and the use of ES cell or an equivalent route.
  • the first method is exemplified in the examples of this application.
  • the transgenic host is preferably a laboratory animal such as a mouse, rat or rabbit or a potential livestock bioreactor animal, including those described above as well as sheep or cattle.
  • a method of obtaining a substance comprising a polypeptide comprising harvesting the substance from a transgenic organism according to the fourth aspect of the invention.
  • the substance is either secreted from the organism for harvesting or is expressed at a specific location by the organism for harvesting.
  • a preferred method for obtaining a substance according to this aspect of the invention is to direct expression of the coding sequence linked to a beta-lactoglobulin promoter and harvest the substance from the mammary gland of mammalian transgenic host organism.
  • the mammal is a non-human mammal, for example a mouse or a sheep.
  • FIGURE 1 shows various examples of a secondary transgene according to the invention including the relationship between the transgene and the flanking sequences .
  • the open box at the top of the Figure (designated AATB) represents the primary transgene.
  • FIGURE 2 shows a restriction map of the transgene locus of mouse line AATB 46.2.
  • FIGURE 3 shows restriction maps of (a) clone 9 and (b) clone 8 which were isolated from the cosmid library from an animal of line AATB 46.2.
  • FIGURE 4 shows a diagram of the Lambda DASH II vector (Stratagene) .
  • FIGURE 5 shows the position of Notl and Srfl restriction sites: (a) in clone 8 and (b) in clone 9.
  • FIGURE 6 shows a diagrammatic construction of the secondary transgene TAB from clones 8 and 9.
  • FIGURE 7 is a graphic representation of AAT expression level from two studies of the transgenic mice containing the primary transgene AATB and one study of transgenic mice containing the secondary transgene TAB .
  • FIGURES 8(a), 8(b) and 8(c) show the construction of a "high expressing" vector (named MV) which contains the 5' and the 3' flanking sequences from the plasmid TAB (pTAB) , deposited at NCIMB as deposit no. 40806.
  • the MV vector can be used to prepare a plasmid for a high expressing transgene (either from that plasmid or from any other genetic construction following integration of plasmid sequences therewith) .
  • FIGURE 9 shows the construct MV-BLG-CAT in which the BLG-CAT sequences were inserted between the 5' and 3 ' MV sequences.
  • FIGURE 10 shows the results of experiments to determine the expression of MV-BLG-CAT constructs in HC-11 cells. The level of expression is measured by the %CAT conversion.
  • FIGURE 11 shows the construct MV-SV40-CAT.
  • FIGURE 12 shows the results of experiments to determine the expression of MV-SV40-CAT constructs in BHK cells .
  • the level of expression is measured by the %CAT conversion.
  • the transgenic mouse line designated AATB 46.2 harbours the transgene designated AATB.
  • the transgene encodes human AAT.
  • the expression of this protein in the mammary gland of the transgenic mice is driven by the ovine BLG promoter.
  • This transgenic mouse line was constructed as described in WO 90/05188 on pages 12 to 23.
  • the expression level of AAT from the line AATB 46.2 was observed as stable through several generations and ranged from 6-8 mg/ml.
  • the locus of AATB 46.2 was mapped using a number of restriction endonucleases using techniques known in the art .
  • the transgenes were found to be in a perfect head to tail array.
  • a restriction map of the locus is given in Figure 2.
  • the number of copies of the transgene in the line was approximately eight.
  • the junction fragments detected using a number of restriction endonucleases and probes are represented with thick lines. The size of the junction fragment is given above each.
  • transgenic mouse line AATB 46.2 was cloned by first constructing a cosmid library of DNA isolated from these mice in the cosmid vector Super COS
  • the cosmid library was constructed using liver DNA from an animal of line AATB 46.2. The DNA was partially digested with the enzyme Ndell and fractionated on a sucrose gradient. Fractions containing fragments of
  • the library was screened using the ovine 5' -BLG sequences.
  • the enzyme Notl was used to liberate the AATB transgene from the pPoly-III-I vector sequences prior to micro-injection. This enzyme produces a 5' overhang of four base pairs at the transgene ends. Analysis of the clones demonstrated that the Notl site had been regenerated between the transgenes within the array. One of the clones included the murine sequences of the 5' end of the array and the other included the murine sequences from the 3 ' end of the array. Both clones were found to harbour a maximum of 5kb of murine sequences . The remainder of the insert sequences were those of the AATB transgene array.
  • Both of the clones 8 and 9 generated Notl fragments of approximately 15 kb .
  • the large Notl fragment from clone 9 encompassed the entire AATB transgene plus all of the murine flanking DNA within the insert. It spanned from the Notl site of the cosmid vector cloning region to the first Notl site within the transgene array. This was identified as the 5' junction fragment of the locus with respect to the 5' -3' orientation of the AATB transgenes within the array. Sequencing across the chromosomal /transgene junction demonstrated that the AATB transgene at the 5' end of the transgene array was intact. No digestion of the transgene end had occurred prior to integration into the genome.
  • the large Notl fragment of clone 8 was similar. It encompassed the AATB transgene from the 3 ' end of the transgene array and the 3 ' flanking murine DNA of the AATB 46.2 locus. This fragment spanned from the most 3' Notl site in the array to the Notl site in the cosmid vector cloning region. Sequencing across the chromosomal /transgene junction in this clone demonstrated that 50bp of the AATB sequences had been removed from the end of the 3' transgene of the array before integration into the genome . This was the only perceptible damage that had occurred within the array.
  • the secondary transgene was constructed from clone 8 and clone 9 and cloned in Stratagene 's Lambda DASH II phage vector.
  • a diagram of this vector is given in Figure 4.
  • the vector accommodates an insert of 14 to 20kb in size.
  • Clones 8 and 9 are those described above in Example 3. Both clone 8 and clone 9 were digested with the restriction enzymes Srfl and Notl. The junction fragments produced by this digest were isolated from both clones.
  • Figure 5 shows the position of Notl and Srfl restriction sites for clones 8 and 9. The fragments which were isolated and purified from the digests for the construction of the secondary transgene are underlined and the respective sizes of the fragments are given.
  • the clone 9, Notl-Srfl junction fragment spanned from the Notl site of the cosmid vector to the Srfl site in the BLG sequence of the AATB transgene .
  • the fragment was 8.7 kb in size and contained all of the murine genomic DNA from clone 9.
  • the clone 8 Notl -Srfl junction fragment spanned from the Srfl site in the BLG sequences of the transgene to the Notl site of the cosmid vector.
  • the fragment was llkb in size and contained the AAT coding sequences of the AATB transgene and all of the murine genomic DNA of clone 8.
  • the secondary transgene has both the 5 ' murine flanking DNA from clone 9 and the 3 ' murine flanking DNA from clone 8. Between the 5' and 3' flanking genomic sequences is a single copy of the AATB transgene. This copy is missing 50bp from the 3' end of the AATB transgene . These sequences are within the non- transcribed 3' sequences of AATB.
  • the secondary transgene also harbours the T3 and T7 primer sites from the cosmid vector. The secondary transgene has Notl ends and these were used to clone the transgene into the Lambda DASH II vector. A small number of recombinant phages were obtained from the ligation.
  • plaques were screened using a 3' AATB probe and a 5' AATB probe. All but one of the plaques hybridised to both probes indicating that the AATB sequences from clone 9 and clone 8 must have been present within the inserts.
  • the secondary transgene insert was purified from the lambda vector sequences following Notl digestion of the clone and was subsequently re-cloned into the pBluescript vector (Stratagene) to generated pTAB. This plasmid was deposited at NCIMB [NCIMB 40806] .
  • the insert from pTAB was excised by Notl digestion and the injection fragment purified by gel electrophoresis.
  • 12 secondary transgenic mouse lines were produced by pronuclear injection of newly fertilized mouse embryos. Construction of these secondary transgenic animals, including injection of DNA and identification of transgenic individuals was as described in WO 90/01588, pages 35 to 46. The transgenic animals were detected using both PCR and Southern blotting procedures. The expression levels of the human protein AAT in the milk of lactating secondary transgenic females was measured in the first produced transgenic animals (the G 0 generation) and in the subsequent generations (G 17 G 2 , etc.).
  • Example 6 above is of a secondary TAB transgene, which includes the genomic flanking sequences from the 46.2 locus. The comparison of expression levels are given in
  • FIG. 7 This compares the average level of expression of AAT in individually generated transgenic mouse lines for the Archibald Study, the Carver Study and the study with the secondary transgene (TAB) .
  • TAB mice In the TAB mice, not only are more mice expressing higher levels of AAT but maximal levels of expression in the TAB group of mice is approximately 3 fold of that observed in either of the other mice studies .
  • Construction of MV Vector Figures 8(a), 8(b) and 8(c) show the construction of a
  • MV "high expressing" vector
  • pTAB plasmid TAB
  • the MV vector can be used to prepare a plasmid for a high expressing transgene (either from that plasmid or from any other genetic construction following integration of plasmid sequences therewith) .
  • pBluescript is referred to as SK.
  • (1) is the cloning of the Not-Sal 4kb fragment into pBlueSK at the appropriate Not and Sal sites.
  • the Sal-Sal 0.6kb fragment is cloned into the SK4.0 Sal site and the orientation checked under item (2) .
  • Figure 8 (b) shows the third step of partial digestion to delete the middle Sal site to generate SK 4.6D.
  • the ligation of murine 5' and 3' flanking sequences is described as Notl and Sail digested flanking clone to release inserts, then ligating the result into pBlueSK Notl site to generate SK 9.6.
  • the modification of the clone vector is described.
  • the construct MV-BLG-CAT was generated using the MV vector (see Figure 8c) and a BLG-CAT hybrid gene using standard recombinant DNA techniques.
  • the BLG-CAT gene comprises a 406kb beta-lactoglobulin (BLG) promoter element linked to the bacterial chloramphenicol acetylase gene (CAT) and has been previously described (Webster et al Cell Biol . Res . 41 11-15 (1995)).
  • the CAT sequences provide a reporter gene whose expression can be conveniently be quantitated.
  • the BLG-CAT sequences were inserted between the 5' and 3' MV sequences as illustrated in Figure 9.
  • the Notl fragment released from the BLG-CAT plasmid was cloned into the Smal restriction site of the MV vector to generate MV-BLG-CAT which was amplified by standard bacterial culture and the plasmid subsequently purified on a CsCl gradient.
  • This construct was compared to that of the original BLG-CAT gene in transient and stable transfections in cell culture using the mouse mammary cell line HC-11.
  • the HC-11 cells were seeded in RPMI 1640 medium and incubated until they were 70% confluent. At this stage, the medium was changed and fresh medium was added.
  • a mix comprising 20 ⁇ g of test DNA, 3 ⁇ g of a plasmid containing the neomycin phosphotransferase gene, 62 ⁇ l of 2M CaCl 2 , 0.5ml distilled water and 0.5 ml of 2X Hepes buffer was made up and 1.0 ml of this mixture was added to the cultured cells. The cells and DNA/CaCl 2 mix were then incubated overnight and after this the culture was split in half.
  • transfected culture was incubated for a further 48 hours in medium containing the hormone prolactin to induce transient expression from the BLG promoter.
  • the other half of the culture was incubated until 90% confluent and then the antibiotic G418 added to a final concentration of 200 ⁇ g/ml. G418 selection was continued or a further 2 weeks to select stable transformants .
  • induction medium containing the hormone prolactin was added and the cells incubated for a further 2 days to induce BLG expression. Both transiently-transfected and stably-transfected cell populations were harvested by gentle centrifugation at the end of the culturing.
  • CAT assays were carried out on protein extracts prepared by freeze-thawing the pellet of cells from both transiently- and stably-transfected cultures. The protein concentration in each sample was measured using the BioRad protein assay kit. CAT activity was assayed in a final volume of 100-200 ⁇ l using l-10 ⁇ g of protein extract in 0.25M Tris, pH8.8 with 14 C-chloramphenicol . The reaction was started using 5 ⁇ l of 50mM acetyl CoA incubated at 37°C for up to 3 hours and then the reaction was stopped using an equal volume of ethyl acetate.
  • MV sequences to enhance expression from an SV40 enhancer promoter-CAT construct in fibroblast cells
  • the above experiment shows that incorporation of MV sequences can be used to enhance expression from a BLG- CAT construct in stably transfected mammary gland cells in culture .
  • the construct MV-SV40-CAT was prepared.
  • the SV40-CAT gene comprises the SV40 enhancer promoter linked to the CAT gene and is analogous to the BLG-CAT gene described in Example 9, except that the BLG promoter is now replaced with SV40 regulatory sequences.
  • the construction of MV-SV40-CAT is illustrated in Figure 11.
  • the 1.4kb SV40-CAT sequences were released from the plasmid pB9SV by digestion with Hindlll and Xbal . The "sticky" ends from these fragments were filled in using Klenow polymerase and cloned into the Smal site of the MV vector to generate MV-SV40-CAT.
  • SV40-CAT and MV-SV40-CAT constructs were compared in transient and stable transfections of baby hamster kidney (BHK) fibroblasts .
  • BHK cells were seeded in Dulbecco's medium and incubated until 30-40% confluent and 3 hours prior to transfection the medium was changed.
  • a mix comprising 20 ⁇ g of test DNA (SV40-CAT or MV-SV40-CAT) , 3 ⁇ g of a plasmid encoding the neomycin phosphotransferase gene, 62 ⁇ l of 2M CaCl 2 , 0.5ml of distilled water and 0.5ml of Hepes buffer was made up and 1.0ml of this mixture was added to the cultured cells.
  • the cells and DNA/CaCl 2 mix were then incubated overnight and after this the culture was split in half. For transient transfections the cells were incubated for a further 48 hours and then assayed for CAT expression.
  • FIG. 12 This figure shows the &CAT conversion, which is a measure of the expression level of the SV40-CAT construct, with and without MV sequences. Incorporation of the MV sequences had no effect on the expression levels of the SV40-CAT transgene after transient expression. However, there was an about 4.5-fold improvement in the level of expression when MV-SV40-CAT was compared to SV40-CAT in the stably transfected cell lines. These results show that the MV sequences can enhance the expression of CAT driven by a non-milk protein gene promoter (i.e. SV40) in non-mammary gland cell lines such as BHK fibroblasts.
  • a non-milk protein gene promoter i.e. SV40

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Abstract

L'invention concerne un produit de recombinaison génétique comprenant ue séquence de régulation liée de manière opérationnelle à une séquence de codage de protéine et/ou d'ARN pour diriger son expression, et à l'ADN adjacent d'un locus transgénique fortement exprimé pour produire une expression améliorée de la protéine et/ou de la séquence de codage d'ARN quand le produit de recombinaison est introduit dans un organisme hôte.
PCT/GB1997/002830 1996-10-14 1997-10-14 Expression transgene WO1998016634A1 (fr)

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WO2002016625A2 (fr) 2000-08-25 2002-02-28 Basf Plant Science Gmbh Polynucleotides vegetaux codant de nouvelles proteases prenyle
US6576464B2 (en) 2000-11-27 2003-06-10 Geron Corporation Methods for providing differentiated stem cells

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WO1992011358A1 (fr) * 1990-12-24 1992-07-09 The Agricultural And Food Research Council Expression amelioree dans des organismes transgeniques au moyen d'une seconde sequence transferee
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WO1992011358A1 (fr) * 1990-12-24 1992-07-09 The Agricultural And Food Research Council Expression amelioree dans des organismes transgeniques au moyen d'une seconde sequence transferee
WO1996017946A1 (fr) * 1994-12-06 1996-06-13 Ppl Therapeutics (Scotland) Ltd. Produit de construction de transgenes et son expression amelioree

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DOBIE K W ET AL: "VARIEGATED TRANSGENE EXPRESSION IN MOUSE MAMMARY-GLAND IS DETERMINED BY THE TRANSGENE INTEGRATION LOCUS", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, (25 JUN 1996) VOL. 93, NO. 13, PP. 6659-6664. ISSN: 0027-8424., XP002053858 *
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002016625A2 (fr) 2000-08-25 2002-02-28 Basf Plant Science Gmbh Polynucleotides vegetaux codant de nouvelles proteases prenyle
US6576464B2 (en) 2000-11-27 2003-06-10 Geron Corporation Methods for providing differentiated stem cells
US8426198B2 (en) 2000-11-27 2013-04-23 Geron Corporation In vitro differentiated cell and human embryonic stem cell population
US9023645B2 (en) 2000-11-27 2015-05-05 Asterias Biotherapeutics, Inc. Isolated in vitro cell population comprising primate pluripotent stem cells containing a nucleic acid construct and differentiated progeny of the pluripotent stem cells

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