CA2108564A1

CA2108564A1 - Gene expression cassette containing non-coding sequence of growth hormone gene

Info

Publication number: CA2108564A1
Application number: CA002108564A
Authority: CA
Inventors: Kevin A. Ward; Colin D. Nancarrow; Alan G. Brownlee
Original assignee: Individual
Current assignee: Commonwealth Scientific and Industrial Research Organization CSIRO
Priority date: 1991-04-16
Filing date: 1992-04-13
Publication date: 1992-10-17
Also published as: WO1992018635A1; ZA922761B; NZ242370A

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

W0 92/18635 ~ pcr/Aus2/ool64 GENE EXPRESSION CASSEl~E CONTAINING NON-CODING SECIUENCE OF GROWrH 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.
BACl~GROUND 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 expres~ion cas~ette consists of two co~ponents:-a regulatory element and a non-coding sequence from the growth hormone gene.
SUM~A~Y OF ~HE PRESENT INVENTION
Accordingly, in a first aspect the present invention consists in a genetic expression cassette fox use in obtaining expression of a cDNA se~uence 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 s WO92/18635 ~6 4 PCT/Aug2/ool 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 in~ention 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 thurinqiensis. 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 ~igure l.
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 i~ ovine or bovine.
E~ ~E~CRIPTION OF THE INVENTION
In order that the nature of the pre9ent invention may be more clearly understood, preferred forms thereof will now b~ descxibed with reference to the following examples ~nd figuxe8 in whichs-Figure l ~hows the nucleotide sequence of the expression c~ssette of the present invention;
Figure 2 shows the sequence of MTCEl0;
Figure ~ shows the sequence of MTCR7;

WO92/18635 210 8 ~ 6 ~ PCl/AU92/00l~
,~ . . .,;, _ 3 -Figure 4 shows the sequence of MTCEK1;
Figure 5 shows the sequence of MTAceA2;
Figure 6 shows the sequence of MTAceB2;
Figure 7 shows the se~uence of MTAceABll; and Figure 8 shows levels of radiolabelled cysteine in transgenic mice containing MTCERl ( - ) and in control mice (~ ). The arrow shows the position of cysteic acid.
Initially, a number of gene arrangements for expression of the cysR gene in murine L-cells were trialled. The trialled constructs were as follows:-pMTCK7 - sheep metallothionein-Ia gene promoter -~y~ - exon 5 of sheep growth hormone.
pNTCK8 - sheep metallothionein-Ia promoter - exon 1 sheep growth hormone - cysR - exon 5 sheep growth hormone.
pMTCKll - sheep metallothionein-Ia promoter - cysK -whole sheep growth hormone.
p~TCK12 - sheep metallothionein-Ia - exon 1 sheep growth hormone - ~y~ - 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.
TA~LE 1 ~; O-Acetyl~erine SulfhYdrYlase Act~vity ln Transfected ~urlne L-~ells Using Variou9 c~K Genes ~QLQ ~æyme Activity ~n~oles cysteine produced/mg protein/30 min) pNTCR7 1350 ~ 24 pMTCR8 510 1 13 p~TCRll 162 + 17 pMTCR12 159 1 6 - 35 (values represent the means of two determinations) ':

~, , W092/18635 ~ PCT/AU92/001 c~

As can seen from these results exon 5 of the growth hormone gene of sheep is required for optimum expres~ion 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 cysR aenes of E. coli in transqenic 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 sulfhydr~lase protein.
The expression cassette of the present invention was produced using methods well known in the^art. Briefly this involves the steps of:
1. I~ola~ion and cloning of the sheep metallothionein-Ia promoter sequence.

2. I~olation and modlfication of the bacterial coding ~equence and ~usLon to the bact~rial coding sequence.

3. Fusion of exon 5 of the sheep growth hormone gene to the metallo~hionein ~romoter/b~cterial coding sequence complex.

WO92/18635 21 0 ~ ~ 6 ~ PCT/A~92/~164 .
In order to provide further details on constructlon 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 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 ceding sequence and associated 5' and 3I 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 M13 and the clones encompassing the bacterial initiation codon and the bacterial stop codon were used for site-directed mutagenesis to introduce a Bam H1 site at the 5' end of the coding seguence 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 3am Hl - Sau 3A
fragmen~ o~ DNA was then excLsed from this placmid and inserted into a fiim~larly digested sample of the plasmid containing the metallothionein-Ia sequence.
Step 3.
; ~he pla~mld containing the metallothlonein-Is promoter-cs~E coding se~uence was digested with Pvu II
(~d~acent to the introduced Sau 3A 8 ite) and to this was ligated a blunt-ended P~t 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.

. .

W092/18635 PCT/AU~2/001~
! :'`

a~ 6~ -6 GENE DETAILS
Gene 1 (~T OE lO) 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 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 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 seyuence, and the growth hormone exon S 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 (~TCX7) This gene consists of the sheep metallothionein-Ia gene promoter sequence ~oined to the coding sequence of the ~s9~ shl~-~ll Çy~ gene at a unique Sal 1 restriction enzyme site. This se~uence was then joined to the 3' sequence of exon 5 of the sheep g owth hormone gene. Minor seguence modification of the cysR gene in the vicinity of the initi~tion codon was made by site-directed mutagenesis using a synthetic oligonucleotide. The metallothionein promoter replaces all regulatory seguences located 5' to the ~y~ codlng sequence, and the sheep growth hormone exon 5 replnces all untranslated sequence located 3' to the Y~ coding sequence. The size of the gene is appxoximately 3750 ba8e psirs in length, of which 2957 b~e p~ir~ have been sequenced. The sequence of gene 2 i9 ~hown in Figure 3.
Bene 3 (~TC~gl) This gene consists of a fusion of genes 1 and 2 to ;

. ~ .
. , , WO92/18635 210 8 5 ~ ll PCT/AU92/001~

create a single DNA sequence that encodes both the serine transacetylase and the O-acetylserine sulfhydrylase enzymes. Each coding seguence is separately regulated by its own adjacent sheep metallothionein-Ia gene promoter S 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 glyoxylate 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 lS 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 ~
Gene 4 (~TAceA2) ~his gene consists of the sheep metallothionein-Ia gene promoter 8equence ~oined to the coding sequence of the EscherichLs coll ~Q~ gene at a unique BamH1 restriction enzyme 81te. Thi8 sequence was then ~oined to the 3' sequence of exon 5 of the 8heep growth hormone gene. ~inor sequence modification ln the ~icinity of the initiation and ~top codons of the bacter~al ace~ gene were made by 81~e-directed mutagenesis u8ing 8ynthetic ollgonucleotides. The metallothionein promoter replaces all regulatory 8equence~ located S' to the 3~ gene coding sequence, and the growth hormone exon 5 sequence !- .

W092/18635 ~6 4 Pcr/Aug2/~l~

replaces all untranslated ~equences located 3' to the aceA
gene coding sequence. The gene is approxlmately 3580 base pairs in length, of which 2827 nucleotides have been sequenced. The sequence of gene ~ 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 ace~ gena at a unique Sal 1 restriction enzyme site. This seguence was then joined to the 3I 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 ha~e been sequenced~ The sequence of gene S is shown in figure 6.
Gene 6 (MTAceABl) This gene consists of a fusion of genes l and 2 to create a single DNA sequence that encod~s both the isocitrate lya8e and the malate synthase enzymes. Each coding sequence is separately xegulated by it8 own ad~acent sheep metallothionein-Ia gene promoter sequence, ~nd each coding seguence i8 separately followed by the 3' sequence of exon 5 of the sheep growth hormone gene. The gene i8 appxoximately 75SO b~se paix8 ln size, of which S784 nucleotide~ have been sequenced. ~he s~quence of gene 6 i8 shown in Figure 7.
R~G~LA~ION QP TE~ OE NES
Regulatlon in Cultured Cells Genes l to 6 have been transfected into mouse L-cells W092t;8635 21 ~ ~ 5 ~ 4 PCT/AU92/00164 _ g _ in culture to produce stably transformed cell line~. The expression of each gene was measured by:
1. Northern blot analysis of extracted RNA.
2. Enzyme assay of cell extracts.
An RNA tran~cript of the expected size was detected in RNA extracted from each cell line, using a probe ~pecific for the appropriate coding ~equence of each gene. The intensity of the hybridisation increased when cells were grown in a medium containing 10 uN 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 transacetyla6e, 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 MTCER1 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 thls cell line were incubated with the substrates serlne and H2S, sub~tantial quantities of cysteine were produced, evldence that the entire biochemical p~thway i~ operational in these cells.
Similarly, cell extracts prepared from the cells containing the fusion gene M~AceABl contained both i~ocitr~te lya8e and malate ~ynthase enzymQ activities, indic~ting that both coding sequences within the fusion ~ene were transcribed and translated.
~pression in Transgenic llice Genes 1 to 6 were each transferred to transgenic mice .

.

WO92/18635 6 ~ PCT/AU92/001~

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.

Expression of MTCE10 and MTCK7 in transformed mouse L-cells Serine Transacetylase O-acetylserine Sulfhydrylase cells -Zn +Zn -Zn +Zn 15 control 0 0 0 0 MTCEKl120 360 1082 7790 Values are nmoles product formed/mg protein/30 min W092/l8~35 21 0 3 ~ ~ ~ PCT/AU92/00l~

.

Activity of serine transacetylase (SAT) and O-acetylserine sulphydrylase (OAS) in tissue extracts prepared from transgenic mice. CR7-26 contains the gene pMTC~7, CE10-29 contains pMTCEl0 and CERl-28 and CERl-8 contains pMTCER1.
Specific activity is measured as nmoles substrate utilised (SAT) or product formed (OAS/30 min/mg protein.

MOUSE LINE ORGAN SAT OAS
CR7-26 Intestine - 206 Kidney - 352 Liver - 13 CEl0-29 Intestine6,546 Kidney 0 Liver 0 15 CER1-28 Intestine1,161 2,797 Kidney 0 24 Liver 0 3 Brain 16 86 CEK1-8 Intestine4,522 12,778 Xidney 105 128 Liver 9 3 3rain 0 245 ~ 0 158 : S~in 0 329 ; 25 6 295 ,~ :
; .

WO92/18635 PCT/AU92/001~
64 ~ ~

In order to assess the ability of transgenic mice containing the pMTCERl gene to produce cysteine, transgenic mice including this gene and control mice were gi~en 25 mM ZnS04 in their drinking water for a minimum of four days. On the day of the experiment the ZnSO4 was relaced with normal drinking water and 60 min. later 30 - 60 uCi of ~a~5S was administered per os. The mice were sacrificed 60 min. later and intestinal tissue homogenised in a buffered aqueous solution containing 10mM
dithiothreitol. Two volumes of performic acid were then added and the s~lution 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:H2O (10:100:900, pH3.6) at a voltage of 200 Volts for 2 hr. The paper was the cut into 0.5 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.

Expression of MTAceA2 and MTAceB2 in transformed mouse L-cells cell lineisocitrate lyase malate synthase control 0 30 MTAceA2 68 MTAceB2 - 34~3 Value~ are nmoles product/mg protein/20 min WO92/18635 ~ 6 ~ PCT/~U92/001~

_ 13 -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 MTAceAB1 intestine 27.2 ND
liver not detectable 182 kidney 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 lO 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 broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Claims

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 cysE, 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.