AU655815B2 - Gene expression cassette containing non-coding sequence of growth hormone gene - Google Patents

Description

i l~trl~ OPI DATE 17/11/92 APPLN. ID 15771/92 I 11111111111111 AOJP DATE q9/1./92 PCT NUMBER PCT/AU92/00164 I111II 111111111111 I I AU9215771 INTERNATIONA' APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 5 International Publication Number: WO 92/18635 C12N 15/85, 15/60, 15/67 Al (43) International Publication Date: 29 October 1992 (29.10.92) (21) International Application Number: (22) International Filing Date: Priority data: PK 5664 16 April PCT/AU92/00164 13 April 1992 (13.04.92) 1991 (16.04.91) (71) Applicant (for all designated States except US): COMMON- WEALTH SCIENTIFIC AND INDUSTRIAL RE- SEARCH ORGANISATION [AU/AU]; 14 Limestone Avenue, Campbell, ACT 2601 (AU).

(72) Inventors; and Inventors/Applicants (for US only) WARD, Kevin, Alan [AU/AU]; 28 Woodbury Street, Wordford, NSW 2778 NANCARROW, Colin, Douglas [AU/AU]; 47 Chelmsford Avenue, Willoughby, NSW 2068 (AU).

BROWNLEE, Alan, George [AU/AU]; 8/1 Pennant Street, Castle Hill, NSW 2154 (AU).

(74) Agent: F.B. RICE CO; 28A Montague Street, Balmain, NSW 2041 (AU).

(81) Designated States: AT (European patent), AU, BE (European patent), BR, CA, CH (European patent), DE (European patent), DK (European patent), ES (European patent), FR (European patent), GB (European patent), GR (European patent), IT (European patent), JP, LU (European patent), MC (European patent), NL (European prtent), SE (European patent), US.

Published With international search report.

655815 (54)Title: GENE EXPRESSION CASSETTE CONTAINING NON-CODING SEQUENCE OF GROWTH HORMONE

GENE

(57) 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.

WO 92/18635 PCT/AU92/00164 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 high-level 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 "transgenic".

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 polypeptides.

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 WO 92/18635 PCT/AU92/00164 2 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 ,quences of bacterial enzymes, plant chitinases, insecticidal scorpion venom toxin and the insecticidal protein of the bacteria Bacillus thurinriensis. In a preferred embodiment of the present invention the cDNA sequence is selected from the group consisting of cysE, cvsK, 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 Figure 3 shows the sequence of MTCK7;

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r iir 1 WO 92/18635 PCT/AU92/00164

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Figure 4 shows the sequence of MTCEK; Figure 5 shows the sequence of MTAceA2; Figure 6 shows the sequence of MTAceB2; Figure 7 shows the sequence of MTAceAB11; 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 cvsK gene in murine L-cells were trialled. The trialled constructs were as follows:pMTCK7 sheep metallothionein-Ia gene promoter cvsK exon 5 of sheep growth hormone.

pMTCK8 sheep metallothionein-Ia promoter exon 1 sheep growth hormone cysK exon 5 sheep growth hormone.

pMTCK11 sheep metallothionein-Ia promoter cysK whole sheep growth hormone.

pMTCK12 sheep metallothionein-la 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 result, obtained are set out in Table 1.

TABLE 1 I 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 pMTCK11 162 17 pMTCK12 159 6 (values represent the means of two determinations) I I i

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WO 92/18635 PCr/AU92/001(%4 4 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 0-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 0-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-la 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.

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WO 92/18635 PCT/AU92/00164 5 In order to provide further details on construction of the cassette the procedure followed in construction of was as follows: Step 1.

A bacterial plasmid containing the sheep metallothionein-Ia gene was digested with the restriction enzymes Eco RI and BamH1 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 R1 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 M13 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 3Afragment 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-csvE 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 Plasmids containing the correct orientation of the growth hormone sequence were identified by restriction enzyme mapping.

WO 92/18635 PCT/AU92/00164 6 GENE DETAILS Gene 1 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 cvsE 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 in 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 cvsK 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 WO 92/18635 PCT/AU92/00164 7 create a single DNA sequence that encodes both the serine transacetylase and the 0-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 lyvoxylate cycle in transqenic 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 BamH1 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 4 4 PCT/AU92/0016 4 WO 92/18635 WO 92/18635 PCT/AU92/00164 8 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 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' 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 2957 base pairs have been sequenced. The sequence of gene 5 is shown in figure 6.

Gene 6 (MTAceAB1) 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 WQ 92/18635 PC/AU92/00164 9 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 extracit from each of the transformed cell lines are shown in Table 1 (genes 1 3) and Table 4 (genes 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 H 2 S, 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 MTAceAB1 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 transgei.ic mice SUBSTITUTE

SHEET

WO 92/18635 PCT/AU92/00164 10' 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 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 MTCE10 and MTCK7 in transformed mouse L-cells Serine Transacetylase O-acetylserine Sulfhydrylase cells -Zn +Zn -Zn +Zn control 0 0 0 0 1281 2706 MTCK7 38 1367 MTCEK1 120 360 1082 7790 Values are nmoles product formed/mg protein/30 min r WOP 92/18635 PCT/AU92/00164 11 TABLE 3 Activity of serine transacatylase (SAT) and 0-acetylserine suiphycirylase (OAS) in tissue extracts prepared from transgenic mice. CK7-26 contains the gene pMTCK7, CE10-29 contains PMTCE10 and CEK1-28 and CEX1-8 contains pI4TCEK1.

Specific activity is measured as nmoles substrate utilised (SAT) or product formed (OAS/30 min/mg protein.

MOUSE LINE 2 K7-26 CE10-29 CEK1-28 C'EK1-8

ORGAN

Intestine Kidney Liver Intestine Kidney Liver Inteztine Kidney Liver Brain Intestine Kidney Liver Brain

SAT

6,546 0 0 1,161 0 0 16 4,522 105 9

OAS

206 352 13 2,797 2 A 3 86 12,778 245 158 329 295 Skin I_ WO 92/18635 PCT/AU92/00164 12 In order to assess the ability of transgenic mice containing the pMTCEK1 gene to produce cysteine, transgenic mice including this gene and control mice were given 25 mM ZnSO 4 in their drinking water for a minimum of four days. On the day of the experiment the ZnSO 4 was relaced with normal drinking water and 60 min. later 35 60 uCi of Na S was administered per os. The mice were sacrificed 60 min. later and intestinal tissue homogenised in a buffered aqueous solution containing dithiothreitol. Two volumes of performic acid were then added and the solution left at room temperature overnight. The suspension was then extracted with chloroform/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:H 2 0 (10:100:900, pH3.6) at a voltage of 200 Volts for 2 hr. The paper was the cut into cm strips 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 nouse L-cells cell line isocitrate lyase malate synthase control 0 0 MTAceA2 68 MTAceB2 -34.3 Values are nmoles product/mg protein/20 min i 1 1 1 WO 92/18635 PTI'/AU92/00164 13 TABLE Expression of MTAceAB1 in transgenic mice Mouse Tissue Isocitrate Lyase Malate Synthase control intestine not detectable not detectable liver not detectable not detectable kidney not detectable not detectable MTAceABl intestine 27.2 ND liver not detectable 182 kidney not detectable 1.6 Values of isocitrate lyase are nmoles product/mg min, and for malate synthase are picomoles product/mg protein/20 min (x 10 2) 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 bro 'y described. The present embodiments are, therefore, to ne considered in all respects as illustrative and not restrictive.

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Claims (7)

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 15 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 cysE, cVsK, 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

7. A transgenic non-human animal as claimed in claim 6 in which the animal is ovine or bovine. s i r i i; 4 i