WO1992018635A1 - Gene expression cassette containing non-coding sequence of growth hormone gene - Google Patents
Gene expression cassette containing non-coding sequence of growth hormone gene Download PDFInfo
- Publication number
- WO1992018635A1 WO1992018635A1 PCT/AU1992/000164 AU9200164W WO9218635A1 WO 1992018635 A1 WO1992018635 A1 WO 1992018635A1 AU 9200164 W AU9200164 W AU 9200164W WO 9218635 A1 WO9218635 A1 WO 9218635A1
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- WIPO (PCT)
- Prior art keywords
- gene
- sequence
- expression cassette
- growth hormone
- coding sequence
- Prior art date
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- 239000000122 growth hormone Substances 0.000 title claims abstract description 13
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Classifications
-
- 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
- C12N9/10—Transferases (2.)
- C12N9/1025—Acyltransferases (2.3)
- C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
-
- 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
-
- 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
- C12N9/88—Lyases (4.)
-
- 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)
-
- 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
- C12N2830/00—Vector systems having a special element relevant for transcription
- C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
- C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
-
- 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
- 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 expression cassette consists of two components:- a regulatory element and a non-coding sequence from the growth hormone gene.
- the present invention consists in a genetic expression cassette for use in obtaining expression of a cDNA sequence in animal cells, the cassette comprising an inducible promoter and the 3 ' non-coding sequence of exon 5 of the growth hormone gene or a portion thereof, the cDNA sequence being positioned between the inducible promoter and the 3' non-coding sequence of exon 5 of the growth hormone gene.
- the inducible promoter is the immediate upstream nucleotide sequence of the sheep metallothionein-Ia gene.
- the expression cassette of the present invention provides a means for the expression of a wide range of genes in transgenic animals, including the coding sequences of bacterial enzymes, plant chitinases, insecticidal scorpion venom toxin and the insecticidal protein of the bacteria Bacillus thurin ⁇ iensis.
- the cDNA sequence is selected from the group consisting of cysE, cysK, aceA and aceB genes of Escherichia coli and the coding sequences of plant chitinases.
- Genes 1 and 2 each encode single bacterial proteins, gene 1 encoding the protein serine transacetylase and gene 2 encoding the protein O-acetylserine sulfhydrylase.
- Gene 3 is a compound gene constructed from gene 1 and gene 2, and encodes both the serine transacetylase protein and the O-acetylserine sulfhydrylase protein.
- Step 1 A bacterial plasmid containing the sheep metallothionein-Ia gene was digested with the restriction enzymes Eco RI and BamHl and a DNA fragment encoding the promoter region of the gene separated by agarose gel electrophoresis and cloned in the plasmid vector pUC8. Step 2.
- the E. coli genes encoding the enzymes isocitrate lyase and malate synthase have been inserted into the expression cassette.
- Genes 1 and 2 each encode single bacterial proteins, gene 1 encoding the protein isocitrate lyase and gene 2 encoding the protein malate synthase.
- Gene 3 is a compound gene constructed from gene 1 and gene 2, and encodes both the isocitrate lyase and the malate synthase proteins.
- Gene 4 ( * MTAceA2) This gene consists of the sheep metallothionein-Ia gene promoter sequence joined to the coding sequence of the Escherichia coli aceA gene at a unique BamHl restriction enzyme site. This sequence was then joined to the 3' sequence of exon 5 of the sheep growth hormone gene. Minor sequence modification in the vicinity of the initiation and stop codons of the bacterial aceA gene were made by site-directed mutagenesis using synthetic oligonucleotides.
- the metallothionein promoter replaces all regulatory sequences located 5' to the aceA gene coding sequence, and the growth hormone exon 5 sequence replaces all untranslated sequences located 3' to the aceA gene coding sequence.
- the gene is approximately 3580 base pairs in length, of which 2827 nucleotides have been sequenced.
- the sequence of gene 4 is shown in Figure 5.
- Gene 5 (MTAceB2)
- This gene consists of the sheep metallothionein-Ia gene promoter sequence joined to the coding sequence of the Escherichia coli aceB gene at a unique Sal 1 restriction enzyme site. This sequence was then joined to the 3' sequence of exon 5 of the sheep growth hormone gene. .Minor sequence modification of the aceB gene in the vicinity of the initiation codon was made by site-directed mutagenesis using a synthetic oligonucleotide. The metallothionein promoter replaces all regulatory sequences located 5 1 to the aceB coding sequence, and the sheep growth hormone exon 5 sequence replaces all untranslated sequence located 3" to the aceB coding sequence. The size of the gene is approximately 3750 base pairs in length, of which 29 * 57 base pairs have been sequenced. The sequence of gene 5 is shown in figure 6.
- Gene 6 (MTAceABl)
- Genes 1 to 6 have been transfected into mouse L-cells in culture to produce stably transformed cell lines.
- the expression of each gene was measured by:
- RNA transcript of the expected size was detected in RNA extracted from each cell line, using a probe specific for the appropriate coding sequence of each gene.
- the intensity of the hybridisation increased when cells were grown in a medium containing 10 uM zinc sulphate, indicating that the genes were regulated by heavy metals.
- SUBSTITUTE SHEET by the technique of single-cell embryo pronuclear microinjection. Mice containing the new genes were analyzed for expression by extracting mRNA and preparing cell-free supernatants from various tissues including liver, kidney and intestine. As shown in Tables 3 and 5, high levels of activity of the various enzymes were detected in appropriate transgenic mice. Furthermore, the expression of the genes in the intestinal tissues was highly zinc-dependent. TABLE 2
- transgenic mice including this gene and control mice were given 25 mM ZnSO, in their drinking water for a minimum of four days. On the day of the experiment the ZnSO, was relaced with normal drinking water and 60 min. later 30 - 60 uCi of Ha 35S was administered per os. The mice were sacrificed 60 min. later and intestinal tissue homogenised in a buffered aqueous solution containing lOmM dithiothreitol. Two volumes of performic acid were then added and the solution left at room temperature overnight.
- L-cells cell line isocitrate lyase malate synthase control 0 0 MTAceA2 68
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Abstract
The present invention provides a genetic expression cassette for use in obtaining expression of a cDNA sequence in animal cells. The expression cassette comprises an inducible promoter and the 3' non-coding sequence of exon 5 of the growth hormone gene or a portion thereof. The cDNA sequence is inserted between the inducible promoter and the exon 5 of the growth hormone genes.
Description
GENE EXPRESSION CASSETTE CONTAINING NON-CODING SEQUENCE OF GROWTH HORMONE GENE
FIELD OF THE INVENTION
The present invention relates to a gene expression cassette which enables expression of cDNA sequences in animal cells. The expression cassette of the present invention is particularly useful in achieving highrlevel expression of bacterial and/or plant genes in animal cells. BACKGROUND OF THE INVENTION
It is now possible to transfer unique pieces of DNA between organisms in such a way that the transferred material becomes a functional part of the genetic information of the recipient organisms. The animals that are produced by this technique are termed "transgenicA One application of this technology is to transfer biochemical pathways from bacteria to domestic animals in order to increase animal productivity. One difficulty which is frequently encountered in efforts to produce such transgenic animals is the lack, or very low levels of expression of the transferred DNA sequences. The present inventors have developed a genetic expression cassette which provides information for the expression of heterologous genes, in particular bacterial genes, in mammalian cells and in several^tissues of transgenic animals, at levels that provide ready detection of the encoded pol peptides.
The expression cassette consists of two components:- a regulatory element and a non-coding sequence from the growth hormone gene. SUMMARY OF THE PRESENT INVENTION Accordingly, in a first aspect the present invention consists in a genetic expression cassette for use in obtaining expression of a cDNA sequence in animal cells, the cassette comprising an inducible promoter and the 3 ' non-coding sequence of exon 5 of the growth hormone gene or a portion thereof, the cDNA sequence being positioned
between the inducible promoter and the 3' non-coding sequence of exon 5 of the growth hormone gene.
In a preferred embodiment of the present invention the inducible promoter is the immediate upstream nucleotide sequence of the sheep metallothionein-Ia gene. The expression cassette of the present invention provides a means for the expression of a wide range of genes in transgenic animals, including the coding sequences of bacterial enzymes, plant chitinases, insecticidal scorpion venom toxin and the insecticidal protein of the bacteria Bacillus thurinσiensis. In a preferred embodiment of the present invention the cDNA sequence is selected from the group consisting of cysE, cysK, aceA and aceB genes of Escherichia coli and the coding sequences of plant chitinases.
In.yet a further preferred embodiment of the present invention the genetic expression cassette has a sequence substantially as shown in Figure 1.
The* expression cassette of the present invention is useful in obtaining high levels of expression of cDNA sequences in animal cells. Accordingly, in a second aspect the present invention consists in a non-human animal including the genetic expression cassette of the first aspect of the present invention. In a preferred embodiment of this aspect the animal is ovine or bovine. DETAILED DESCRIPTION OF THE INVENTION
In order that the nature of the present invention may be more clearly understood, preferred forms thereof will now be described with reference to the following examples and figures in which:-
Figure 1 shows the nucleotide sequence of the expression cassette of the present invention; Figure 2 shows the sequence of MTCE10; Figure 3 shows the sequence of MTCK7;
Figure 4 shows the sequence of MTCE 1; Figure 5 shows the sequence of MTAceA2; Figure 6 shows the sequence of MTAceB2; Figure 7 shows the sequence of MTAceABll; and Figure 8 shows levels of radiolabelled cysteine in transgenic mice containing MTCEK1 ( ) and in control mice ( ) . The arrow shows the position of cysteic acid.
Initially, a number of gene arrangements for expression of the cysK gene in murine L-cells were trialled. The trialled constructs were as follows:- pMTCK7 - sheep metallothionein-Ia gene promoter - cysK - exon 5 of sheep growth hormone. pMTCK8 - sheep metallothionein-Ia promoter - exon 1 sheep growth hormone - cysK - exon 5 sheep growth hormone. pMTCKll - sheep metallothionein-Ia promoter - cysK - whole sheep growth hormone. pMTCK12 - sheep metallothionein-Ia - exon 1 sheep growth hormone - cysK - exons 2, 3, 4 and 5 sheep growth hormone.
The constructs were transfected into murine L-cells and the O-acetylserine sulfhydrylase activity of the transfected cells measured. The results- obtained are set out in Table 1.
TABLE 1 O-Acetylserine Sulfhydrylase Activity in Transfected Murine L-Cells Using Various cysK Genes Gene Enzyme Activity (nMoles cysteine produced/mg protein/30 min) pMTCK7 1350 ± 24 pMTCK8 510 + 13 pMTCKll 162 ± 17 pMTC 12 159 ± 6
(values represent the means of two determinations)
As can seen from these results exon 5 of the growth hormone gene of sheep is required for optimum expression of genes inserted into the cassette. Other combinations which comprise larger portions of the sheep growth hormone gene are less effective in providing expression.
Two examples of the function of the expression cassette are shown as follows:
1. Expression of the cysE and cysK genes of E. coli in transgenic animals In order to provide a pathway for the biosynthesis of the amino acid cysteine, the coding sequences for the bacterial enzymes serine transacetylase and O-acetylserine sulfhydrylase have been inserted into the expression cassette. Three genes are described. Genes 1 and 2 each encode single bacterial proteins, gene 1 encoding the protein serine transacetylase and gene 2 encoding the protein O-acetylserine sulfhydrylase. Gene 3 is a compound gene constructed from gene 1 and gene 2, and encodes both the serine transacetylase protein and the O-acetylserine sulfhydrylase protein.
The expression cassette of the present invention was produced using methods well known in the^art. Briefly this involves the steps of: 1. Isolation and cloning of the sheep metallothionein-Ia promoter sequence.
2. Isolation and modification of the bacterial coding sequence and fusion to the bacterial coding sequence.
3. Fusion of exon 5 of the sheep growth hormone gene to the metallothionein promoter/bacterial coding sequence complex.
In order to provide further details on construction of the cassette the procedure followed in construction of MTCE10 was as follows: Step 1. A bacterial plasmid containing the sheep metallothionein-Ia gene was digested with the restriction enzymes Eco RI and BamHl and a DNA fragment encoding the promoter region of the gene separated by agarose gel electrophoresis and cloned in the plasmid vector pUC8. Step 2.
The coding sequence and associated 5' and 3' DNA encompassing the cysE gene of Escherichia coil was cloned in the plasmid vector pGEM3 as an Eco Rl fragment excised from a lambda transducing phage containing portion of the E.coil chromosome. Sub-fragments of this insert were then cloned into the bacteriophage Ml3 and the clones encompassing the bacterial initiation codon and the bacterial stop codon were used for site-directed mutagenesis to introduce a Bam HI site at the 5' end of the coding sequence and a Sau 3A site at the 3* end of the gene. The mutagenesis was carried out on single-strand DNA by conventional procedures and the resulting modified DNA used to replace the corresponding DNA fragments in the insert of the original pGEM3 clone. A Bam HI - Sau 3A fragment of DNA was then excised from this plasmid and inserted into a similarly digested sample of the plasmid containing the metallothionein-Ia sequence. Step 3.
The plasmid containing the metallothionein-Ia promoter-csyE coding sequence was digested with Pvu II (adjacent to the introduced Sau 3A site) and to this was ligated a blunt-ended Pst 1 DNA fragment isolated from the sheep growth hormone gene and encompassing exon 5. Plasmids containing the correct orientation of the growth hormone sequence were identified by restriction enzyme mapping.
GENE DETAILS
Gene 1 (MTCE10)
This gene consists of the sheep metallothionein-Ia gene promoter sequence joined to the coding sequence of the Escherichia coli cysE gene at a unique BamHl restriction enzyme site. This sequence was then joined to the 3' sequence of exon 5 of the sheep growth hormone gene. Minor sequence modification in the vicinity of the initiation and stop codons of the bacterial cysE gene were made by site-directed mutagenesis using synthetic oligonucleotides. The metallothionein promoter replaces all regulatory sequences located 5' to the cysE gene coding sequence, and the growth hormone exon 5 sequence replaces all untranslated sequences located 3' to the cysE gene coding sequence. The gene is approximately 3580 base pairs i length, of which 2827 nucleotides have been sequenced. The sequence of gene 1 is shown in Figure 2. Gene 2 (MTCK7)
This' gene consists of the sheep metallothionein-Ia gene promoter sequence joined to the coding sequence of the Escherichia coli cysK gene at a unique Sal 1 restriction enzyme site. This sequence was then joined to the 3' sequence of exon 5 of the sheep growth hormone gene. Minor sequence modification of the cysK gene in the vicinity of the initiation codon was made by site-directed mutagenesis using a synthetic oligonucleotide. The metallothionein promoter replaces all regulatory sequences located 5' to the cysK coding sequence, and the sheep growth hormone exon 5 replaces all untranslated sequence located 3' to the cysK coding sequence. The size of the gene is approximately 3750 base pairs in length, of which 2957 base pairs have been sequenced. The sequence of gene 2 is shown in Figure 3. Gene 3 (MTCEK1) This gene consists of a fusion of genes 1 and 2 to
create a single DNA sequence that encodes both the serine transacetylase and the O-acetylserine sulfhydrylase enzymes. Each coding sequence is separately regulated by its own adjacent sheep metallothionein-Ia gene promoter sequence, and each coding sequence is separately followed by the 3' sequence of exon 5 of the sheep growth hormone gene. The gene is approximately 7550 base pairs in size, of which 5784 nucleotides have been sequenced. The sequence of gene 3 is shown in Figure 4. Example 2. The expression of the σlyoxylate cycle in transgenic animals
In order to provide the enzymes needed for the operation of the glyoxylate cycle in transgenic animals, the E. coli genes encoding the enzymes isocitrate lyase and malate synthase have been inserted into the expression cassette..
Three genes are described. Genes 1 and 2 each encode single bacterial proteins, gene 1 encoding the protein isocitrate lyase and gene 2 encoding the protein malate synthase. Gene 3 is a compound gene constructed from gene 1 and gene 2, and encodes both the isocitrate lyase and the malate synthase proteins. GENE DETAILS
Gene 4 (*MTAceA2) This gene consists of the sheep metallothionein-Ia gene promoter sequence joined to the coding sequence of the Escherichia coli aceA gene at a unique BamHl restriction enzyme site. This sequence was then joined to the 3' sequence of exon 5 of the sheep growth hormone gene. Minor sequence modification in the vicinity of the initiation and stop codons of the bacterial aceA gene were made by site-directed mutagenesis using synthetic oligonucleotides. The metallothionein promoter replaces all regulatory sequences located 5' to the aceA gene coding sequence, and the growth hormone exon 5 sequence
replaces all untranslated sequences located 3' to the aceA gene coding sequence. The gene is approximately 3580 base pairs in length, of which 2827 nucleotides have been sequenced. The sequence of gene 4 is shown in Figure 5. Gene 5 (MTAceB2)
This gene consists of the sheep metallothionein-Ia gene promoter sequence joined to the coding sequence of the Escherichia coli aceB gene at a unique Sal 1 restriction enzyme site. This sequence was then joined to the 3' sequence of exon 5 of the sheep growth hormone gene. .Minor sequence modification of the aceB gene in the vicinity of the initiation codon was made by site-directed mutagenesis using a synthetic oligonucleotide. The metallothionein promoter replaces all regulatory sequences located 51 to the aceB coding sequence, and the sheep growth hormone exon 5 sequence replaces all untranslated sequence located 3" to the aceB coding sequence. The size of the gene is approximately 3750 base pairs in length, of which 29*57 base pairs have been sequenced. The sequence of gene 5 is shown in figure 6. Gene 6 (MTAceABl)
This gene consists of a fusion of genes 1 and 2 to create a single DNA sequence that encodes both the isocitrate lyase and the malate synthase enzymes. Each coding sequence is separately regulated by its own adjacent sheep metallothionein-Ia gene promoter sequence, and each coding sequence is separately followed by the 3' sequence of exon 5 of the sheep growth hormone gene. The gene is approximately 7550 base pairs in size, of which 5784 nucleotides have been sequenced. The sequence of gene 6 is shown in Figure 7. REGULATION OF THE GENES
Regulation in Cultured Cells
Genes 1 to 6 have been transfected into mouse L-cells
in culture to produce stably transformed cell lines. The expression of each gene was measured by:
1. Northern blot analysis of extracted RNA.
2. Enzyme assay of cell extracts. An RNA transcript of the expected size was detected in RNA extracted from each cell line, using a probe specific for the appropriate coding sequence of each gene. The intensity of the hybridisation increased when cells were grown in a medium containing 10 uM zinc sulphate, indicating that the genes were regulated by heavy metals.
The results of enzyme assays of cell extracts from each of the transformed cell lines are shown in Table 1 (genes 1 - 3) and Table 4 (genes 4,5). High levels of activity of serine transacetylase, O-acetylserine sulfhydrylase, isocitrate lyase and malate synthase were measured in the appropriate cell extracts, and the enzyme levels were increased when cells were grown in zinc-supplemented growth media. Cell extracts prepared from cells containing the fusion gene MTCEK1 contained both serine transacetylase and O-acetylserine sulfhydrylase enzyme activities, indicating that both coding sequences within the fusion gene were transcribed and translated. Furthermore, when extracts from this cell line were incubated with the substrates serine and H2S, substantial quantities of cysteine were produced, evidence that the entire biochemical pathway is operational in these cells. Similarly, cell extracts prepared from the cells containing the fusion gene MTAceABl contained both isocitrate lyase and malate synthase enzyme activities, indicating that both coding sequences within the fusion gene were transcribed and translated. Expression in Transgenic Mice Genes 1 to 6 were each transferred to transgenic mice
SUBSTITUTE SHEET
by the technique of single-cell embryo pronuclear microinjection. Mice containing the new genes were analyzed for expression by extracting mRNA and preparing cell-free supernatants from various tissues including liver, kidney and intestine. As shown in Tables 3 and 5, high levels of activity of the various enzymes were detected in appropriate transgenic mice. Furthermore, the expression of the genes in the intestinal tissues was highly zinc-dependent. TABLE 2
Expression of MTCEIO and MTCK7 in transformed mouse L-cells Serine Transacetylase O-acetylserine
Sulfhydrylase
-Zn +Zn 0 0
38 1367
1082 7790 Values are nmoles product formed/mg protein/30 min
TABLE 3
Activity of serine transacetylase (SAT) and O-acetylserine sulphydrylase (OAS) in tissue extracts prepared from transgenic mice. CK7-26 contains the gene pMTCK7, CE10-29 contains pMTCElO and CEK1-28 and CEK1-8 contains pMTCEKl. Specific activity is measured as nmoles substrate utilised (SAT) or product formed (OAS/30 min/ g protein.
In order to assess the ability of transgenic mice containing the pMTCEKl gene to produce cysteine, transgenic mice including this gene and control mice were given 25 mM ZnSO, in their drinking water for a minimum of four days. On the day of the experiment the ZnSO, was relaced with normal drinking water and 60 min. later 30 - 60 uCi of Ha 35S was administered per os. The mice were sacrificed 60 min. later and intestinal tissue homogenised in a buffered aqueous solution containing lOmM dithiothreitol. Two volumes of performic acid were then added and the solution left at room temperature overnight. The suspensio was then extracted with chlσroform/methanol by conventional means and the aqueous layer concentrated by evaporation. Aliquots of the solution were then placed on Whatman 3mm filter paper and subjected to electrophoresis in a solution of pyridine:acetic acid:H20 (10:100:900, pH3.6) at a voltage of 200 Volts for 2 hr. The paper was the cut into
0.5 cm s-trips and radioactivity counted in a scintillation counter under standard conditions. The results are shown in Figure 8. As can be seen from these results the transgenic mice were able to synthesise radiolabelled cysteine from the administered sodium sulphide in contrast to the control mice. TABLE 4
Expression of MTAceA2 and MTAceB2 in transformed mouse
L-cells cell line isocitrate lyase malate synthase control 0 0 MTAceA2 68
MTAceB2 - 34.3
Values are nmoles product/mg protein/20 min
TABLE 5
Expression of MTAceABl in transgenic mice
Isocitrate Lyase Malate Synthase not detectable not detectable not detectable not detectable not detectable not detectable
27.2 ND not detectable 182
not detectable 1.6 Values of isocitrate lyase are nmoles product/mg protein/20 min, and for malate synthase are picomoles product/mg protein/20 min (x 10 )
It will be appreciated by persons* skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.
Claims
1. A genetic expression cassette for use in obtaining expression of a cDNA sequence in animal cells, the cassette comprising an inducible promoter and the 3' non-coding sequence of exon 5 of the growth hormone gene or a portion thereof, the cDNA sequence being positioned between the inducible promoter and the 3' non-coding sequence of exon 5 of the growth hormone gene.
2. A genetic expression cassette as claimed in claim 1 in which the inducible promoter is the immediate upstream nucleotide sequence of the sheep metallothionein-Ia gene.
3. A genetic expression cassette as claimed in claim 1 or claim 2 in which the cDNA codes for a bacterial enzyme, plant chitinase, insecticidal scorpion vermon toxin or the insecticidal protein of Bacillus thuringiensis.
4. A genetic expression cassette as claimed in claim 3 in which the cDNA sequence is selected from the group consisting of cysEf cysK, aceA and aceB genes of Escherichia coli.
5. A genetic expression cassette as claimed in claim 1 in which the expression cassette has a sequence substantially as shown in Figure 1.
6. A transgenic non-human animal including the genetic expression cassette as claimed in any one of claims 1 to 5.
7. A transgenic non-human animal as claimed in claim 6 in which the animal is ovine or bovine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU15771/92A AU655815B2 (en) | 1991-04-16 | 1992-04-13 | Gene expression cassette containing non-coding sequence of growth hormone gene |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AUPK566491 | 1991-04-16 | ||
| AUPK5664 | 1991-04-16 |
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| PCT/AU1992/000164 WO1992018635A1 (en) | 1991-04-16 | 1992-04-13 | Gene expression cassette containing non-coding sequence of growth hormone gene |
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| NZ (1) | NZ242370A (en) |
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| ZA (1) | ZA922761B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0742285A1 (en) * | 1995-05-10 | 1996-11-13 | Rijksuniversiteit Leiden | Method of isolating exonic gene segments of eukaryotic genes, exon trapping vectors for use therein |
| WO2001025447A1 (en) * | 1999-10-04 | 2001-04-12 | Ajinomoto Co., Inc. | Thermophilic amino acid biosynthesis system enzyme gene of thermotolerant coryneform bacterium |
-
1992
- 1992-04-13 WO PCT/AU1992/000164 patent/WO1992018635A1/en active Application Filing
- 1992-04-13 CA CA002108564A patent/CA2108564A1/en not_active Abandoned
- 1992-04-15 ZA ZA922761A patent/ZA922761B/en unknown
- 1992-04-15 NZ NZ242370A patent/NZ242370A/en not_active IP Right Cessation
Non-Patent Citations (5)
| Title |
|---|
| BYRNE, C.R. et al., AUSTRALIAN JOURNAL OF BIOLOGICAL SCIENCES, Volume 40, No. 4, 1987, "The Isolation and Characterization of the Ovine Growth Hormone Gene", see pages 459-468. * |
| CURATOLA, A.M. and C. BASILICO, MOLECULAR AND CELLULAR BIOLOGY, Volume 10, No. 6, June 1980, (American Society for Microbiology), "Expression of the K-fgf Proto-Oncogene is Controlled by 3 1 Regulatory Elements Which Are Specific for Embryonal Carcinoma Cells", see pages 2575-2483. * |
| GUTKIND, J.S. et al., MOLECULAR AND CELLULAR BIOLOGY, Volume 11, No. 3, March 1991, (American Society for Microbiology), "A Novel c-fgr Exon Utilized in Epstein-Barr Virus-Infected B Lymphocytes but Not in Normal Monocytes", see pages 1500-1507. * |
| HAMPSON, R.K. et al., MOLECULAR AND CELLULAR BIOLOGY, Volume 9, No. 4, April 1989 (American Society for Microbiology), "Alternative Processing of Bovine Growth Hormone mRNA is Influenced by Downstream Exon Sequences", see pages 1604-1610. * |
| ORIAN, J.M. et al., NUCLEIC ACIDS RESERACH, Volume 16, No. 18, 1988, (IRL Press Limited), "Cloning and sequencing of the ovine growth hormone gene", see page 9046. * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0742285A1 (en) * | 1995-05-10 | 1996-11-13 | Rijksuniversiteit Leiden | Method of isolating exonic gene segments of eukaryotic genes, exon trapping vectors for use therein |
| WO2001025447A1 (en) * | 1999-10-04 | 2001-04-12 | Ajinomoto Co., Inc. | Thermophilic amino acid biosynthesis system enzyme gene of thermotolerant coryneform bacterium |
| US6995250B1 (en) | 1999-10-04 | 2006-02-07 | Ajinomoto Co., Inc. | Thermophilic amino acid biosynthesis system enzyme gene of thermotolerant coryneform bacterium |
| US7125977B2 (en) | 1999-10-04 | 2006-10-24 | Ajinomoto Co., Inc. | Genes for heat resistant enzymes of amino acid biosynthetic pathway derived from thermophilic coryneform bacteria |
| US7183403B2 (en) | 1999-10-04 | 2007-02-27 | Ajinomoto Co., Inc. | Genes for heat resistant enzymes of amino acid biosynthetic pathway derived from thermophilic coryneform bacteria |
Also Published As
| Publication number | Publication date |
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| ZA922761B (en) | 1992-12-30 |
| NZ242370A (en) | 1993-02-25 |
| CA2108564A1 (en) | 1992-10-17 |
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