WO1998016634A1

WO1998016634A1 - Transgene expression

Info

Publication number: WO1998016634A1
Application number: PCT/GB1997/002830
Authority: WO
Inventors: Anthony John Clark
Original assignee: Biotechnology And Biological Sciences Research Council
Priority date: 1996-10-14
Filing date: 1997-10-14
Publication date: 1998-04-23
Also published as: GB9621383D0; AU4710997A

Abstract

A genetic construct is provided which comprises a regulatory sequence operatively linked to a protein and/or RNA coding sequence to direct its expression and 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.

Description

TR7ANSGENE EXPRESSION

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 .

The use of 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.

Further observations are that some 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) .

Thus, for a highly expressing transgenic line the transgene would have integrated into a permissive site.

In contrast, in a poorly expressing transgenic line, or a silent transgenic line, the transgene would have integrated into a non-permissive site. Using this theory, 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.

According to a first aspect of the invention there is provided 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.

The term "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.

The term "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 . Preferably, 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) .

An example of 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. Most preferably the construct comprises the murine derived flanking DNA of the secondary transgene TAB contained in the cell line deposited under the Accession No. NCIMB 40806 (at the National Collections of Industrial and Marine Bacteria Ltd, on 14 June 1996, E. coli, K12, DH5Q!, containing pTAB) . 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. Examples of 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. Clearly, if 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.

According to a second aspect of the invention, there is provided 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

[Stratagene] , pPoly III-I described in Lathe et al { Gene 57 193-201 (1987)) and Bluescript [Stratagene].

According to a third aspect of the invention there is provided an eukaryotic cell comprising a genetic construct according to any part of the first aspect of the invention.

Most advantageously 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 .

According to a fourth aspect of the invention there is provided 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. Such 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.

According to a fifth aspect of the invention there is provided 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.

According to a sixth aspect of the invention there is provided a method of obtaining a substance comprising a polypeptide, the method comprising harvesting the substance from a transgenic organism according to the fourth aspect of the invention. Again, such methods are commonplace in the art and are well known by the skilled man for example immunoprecipitation and column chromatography. Advantageously, 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. Preferably the mammal is a non-human mammal, for example a mouse or a sheep.

Preferred features for the second and subsequent aspects of the invention are as for the first aspect mutatis mutandis .

The invention will now be illustrated by the following examples which are provided for the purposes of illustration and which refer to the accompanying drawings, in which:

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.

Where not specifically detailed, recombinant DNA and molecular biological procedures were after Maniatis et al

{Molecular Cloning, Cold Spring Harbor (1982)), Sambrook et al (Molecular Cloning Cold Spring Harbor (1989) ) ,

"Recombinant DNA" in Methods in Enzymology 68, ed. R. Wu,

Academic Press (1979) ; "Recombinant DNA Part B" in Methods in Enzymology 100, ed. Wu et al , Academic Press

(1983) ; "Recombinant DNA Part C" in Methods in Enzymology

101, ed. Wu eϋ al , Academic Press (1983) ; and "Guide to

Molecular Cloning Techniques" in Methods in Enzymology

152, ed. S.L. Berger & A.R. Kimmel) , Academic Press (1987) . Unless specifically stated, all chemicals were purchased from BDH Chemicals Ltd, Poole, Dorset, England or the Sigma Chemical Company, Poole, Dorset, England. Unless specifically stated all DNA modifying enzymes and restriction endonucleases were purchased from BCL, Boehringer Mannheim House, Bell Lane, Lewes, East Sussex BN7 1LG, UK. [Abbreviations: bp = base pairs; kb = kilobase pairs; AAT = alphal-antitrypsin; BLG = beta- lactoglobulin; ]

EXAMPLE 1

Mapping of Transgene DNA from Transgenic Mice 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.

EXAMPLE 2

Preparation of A Cosmid Library containing the 46.2 locus

The AATB locus in 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

(Stratagene) . 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

30kb to 50kb in size were pooled and subsequently ligated into the BamHI site of the cosmid vector cloning site.

To ensure representation of the entire mouse genome in the library 800,000 colonies were screened. The library was screened using the ovine 5' -BLG sequences.

EXAMPLE 3

Mapping of Clones From the Cosmid Library

Two clones were isolated from the library established in Example 2 above which contained murine DNA flanking the transgene locus. Comparison of the restriction maps of these clones with the map previously obtained from the genomic DNA demonstrated that the locus had been cloned without rearrangement .

During the construction of the transgenic mice 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. The restriction maps of the two clones are given in Figure 3 (a) and (b) . In Figure 3 (a) , (clone 9) , the restriction fragments which span the chromosomal/transgene junction are represented by thick lines. These fragments hybridise only with probes from the 5' end of the AATB transgene. The size of the fragments was consistent with the 5' hybridising fragments obtained from the genomic mapping study of the locus of AATB 46.2. The insert harboured 4.6 kb of murine flanking DNA.

In Figure 3(b), (clone 8), the restriction fragments which span the chromosomal/transgene junction are also represented by thick lines. The size of the junction fragments of this clone correlated with those identified using 3' AATB probes in the genomic mapping study. The insert harboured approximately 5 kb of murine flanking DNA.

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.

EXAMPLE 4 Construction of The Secondary Transgene From Clones 8 and 9

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. In the Figure the Notl sites which were used to clone the secondary transgene into the vector are shown. 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. These fragments were ligated together in the presence of Notl so that any Notl complementary end ligations that had occurred would be destroyed. Thus, the predominant ligation product was that of the two fragments ligated at the blunt Srfl site which regenerated the AATB transgene. The ligated fragments were then subjected to gel electrophoresis and the ligation product was purified from an 0.3% agarose gel. The construction of the secondary transgene is diagrammatically shown in Figure 6.

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. 30 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] . EXAMPLE 5

Generation of Secondary Transgenic Animals 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 G₀ generation) and in the subsequent generations (G₁₇ G₂, etc.).

EXAMPLE 6

Expression From The Secondary Transgene in Transgenic Mice Analysis of expression was as described in WO 90/05188, pages 46 to 59. Unlike previous studies of AATB expression in which the flanking sequences were not included in the transgene construct the majority of the females harbouring the secondary transgene gave high levels of expression in the milk. The expression levels and copy numbers of the TAB carrying transgenic mice lines are given in Table 1. This table includes data from both G₀ and G mice. All of the mice of the animal or line designations from Table 1 are of the 12 secondary transgenic mouse lines described above in Example 5 or are derived therefrom. Table 1

In Table 1 the average expression level in mg/ml was calculated from three independently recorded measurements. The standard deviation (σ) from the average expression level is also given. The average expression per copy of the transgene is shown in the final columns.

Only one secondary transgenic founder and her offspring did not express the transgene. All other secondary transgenic females expressed the secondary transgene. More than half the transgenic mice/lines generated expressed human AAT in the milk at concentrations greater than 5g/litre. EXAMPLE 7

Comparison of Expression of AATB as a Primary Transgene and as a Secondary Transgene .

Expression levels of AATB primary transgene and TAB secondary transgenes in mice lines from three studies were compared. The studies were:

1) Carver et al . , Biotechnology. 11, 1263-1270, 1993. 2) Archibald et al . , Proc . Na tl . Acad . Sci . USA, 87 5178-5182, 1990. 3) Results from Table 1 above in Example 7.

The data from Carver et al . and Archibald et al . is of primary AATB transgene. The data from Table 1 from

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

Figure 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) . 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 .

EXAMPLE 8

Construction of MV Vector 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) . In Figures 8 (a) , 8 (b) and 8 (c) , pBluescript is referred to as SK.

In Figure 8(a), the identification of the 5'- and 3'- flanking sequences is shown. The first step under point

(1) is the cloning of the Not-Sal 4kb fragment into pBlueSK at the appropriate Not and Sal sites. Secondly, 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. Fourthly, 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. Under point (5), the modification of the clone vector is described. In the vector SK shown, Sail, Smal and Clal restriction sites exist in the linker region. These sites must be modified in order to remove the Sail site. This is shown schematically in Figure 8 (c) where SK was digested with Sma and Sal, with filing-in then self-ligation again to generate SKD vector. In item (6) , the transfer of the SK9.6 Notl insert into the SKD Notl site is shown which generates SKD9.6. Finally, under item (7), the addition of the linker into SKD9.6 is described. The linker region was designed as Sal-Sma-Bcl-Cla-Sal^* and ligated to SKD9.6 Sal site (Sal^* was modified and this site disappeared after ligation) . The orientation of the linker was subsequently checked. The final vector

SKD9.6V is also named MV. EXAMPLE 9

Use of MV sequence to enhance CAT gene expression in mammary gland cells in culture

An experiment was carried out to determine if MV sequences improved transgene expression in mammary gland cells in culture. In these experiments, the expression of a hybrid construct gene BLG-CAT was compared with and without the incorporation of the flanking MV sequences. The levels of expression were compared in transiently transfected cells (where little or no DNA integration has taken place) to stably transfected cells in which antibiotic selection selects those cells wherein integration into the chromosomal DNA has taken place.

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. In this construct, 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.

The expression of 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 20μg of test DNA, 3μg of a plasmid containing the neomycin phosphotransferase gene, 62μl of 2M CaCl₂, 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/CaCl₂ mix were then incubated overnight and after this the culture was split in half.

One half of the 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 200μg/ml. G418 selection was continued or a further 2 weeks to select stable transformants . At the end of the selection procedure, 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 100-200μl using l-10μg of protein extract in 0.25M Tris, pH8.8 with ¹⁴C-chloramphenicol . The reaction was started using 5μl 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. Samples were freeze-dried, resuspended in 30μl of ethyl acetate, spotted on TLC plates and run in chloroform :methanol 95:5, to separate the acetylated derivatives. After separation, the degree of acetylation in terms of the %CAT conversion was estimated using a Phospholmager and Image-Quant software.

The results from these experiments are tabulated in Figure 10. This figure shows the %CAT conversion which is a measure of the expression level of the BLG-CAT construct with and without the MV sequence .

Incorporation of the MV sequences had no effect on the expression levels of the BLG-CAT transgene after transient expression. However, there was about a 15-fold improvement in the level of expression when MV-BLG-CAT was compared to BLG-CAT in the stably transfected cell lines. These results suggest that for the MV sequence to exert their effects they must be incorporated in the chromosomal sequences.

EXAMPLE 10

Use of 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 . To test whether the MV sequence would function to enhance expression from other promoters in other cell types, 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.

Expression of SV40-CAT and MV-SV40-CAT constructs were compared in transient and stable transfections of baby hamster kidney (BHK) fibroblasts . The 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 20μg of test DNA (SV40-CAT or MV-SV40-CAT) , 3μg of a plasmid encoding the neomycin phosphotransferase gene, 62μl of 2M CaCl₂, 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/CaCl₂ 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.

The other half of the culture was incubated until 90% confluent and the antibiotic G418 added to a final concentration of 200μg/ml. G418 selection was continued for a further 2-3 weeks to select stable transformants. Both transiently transfected and stably transfected cell populations were harvested by gentle centrifugation at the end of the culturing and CAT expression was measured as described in Example 9 above.

The results from the these experiments are tabulated in Figure 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.

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Fora 3?/9 (second and last page)

Claims

1. A genetic construct comprising sufficient regulatory sequence operatively linked to a protein and/or RNA j 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.

2. A genetic construct as claimed in claim 1 wherein the flanking DNA from the highly expressed transgenic loci comprises both 5' and 3' flanking sequences.

3. A genetic construct as claimed in claim 2 wherein the 5' flanking sequence is located on the 5' side of the coding sequence and the 3' flanking sequence is located on the 3' side of the coding sequence.

4. A genetic construct as claimed in any one of claims 1 to 3 wherein the flanking sequences are sufficient flanking DNA from the flanking DNA of the secondary transgene TAB contained in the cell line deposited under the Accession No. NCIMB 40806.

5. A genetic construct as claimed in claim 4 wherein the flanking DNA comprises all or part of the 5' flanking sequence, all or part of the 3' flanking sequence or all or part of both the 5' and the 3' flanking sequences from 0 the secondary transgene TAB contained in the cell line deposited under the Accession number NCIMB 40806.

6. A genetic construct comprising sufficient regulatory sequences operatively linked to a protein to direct its expression and the murine derived flanking DNA of the secondary transgene TAB contained in the cell line deposited under the Accession number NCIMB 40806.

7. A genetic construct as claimed in any one of the preceding wherein the protein and/or RNA coding sequence is human derived.

8. A genetic construct as claimed in claim 7 wherein the protein and/or RNA coding sequence is human alpha 1- antitrypsin.

9. A genetic construct as claimed in any one of the preceding claims wherein the regulatory sequence comprises the BLG promoter.

10. A vector comprising a genetic construct as claimed in any one of the preceding claims.

11. A vector as claimed in claim 10 which is the vector Lambda Dash II, pPoly III-I or Bluescript .

12. A eukaryotic or prokaryotic cell comprising a genetic construct as claimed in any one of claims 1 to 9.

13. A eukaryotic cell as claimed in claim 12 which is any mammalian cell, preferably a murine or ovine cell.

14. A transgenic organism comprising a genetic construct as claimed in any one of claims 1 to 9 integrated into its genome.

15. A transgenic organism capable of improved expression of a protein and/or RNA coding sequence which it carried, wherein the protein and/or RNA coding sequence transgene is part of a genetic construct as claimed in any one of claims 1 to 9.

16. A transgenic organism as claimed in claim 14 or claim 15 which is any mammal, preferably a mouse or a sheep.

17. A method of providing improved expression of a protein and/or RNA coding sequence from a transgenic host organism, the method comprising introducing in the host organism a genetic construct as claimed in any one of claims 1 to 9.

18. A method of obtaining a substance comprising a polypeptide, the method comprising harvesting the substance from a transgenic organism as claimed in claim 15 or claim 16.

19. A method as claimed in claim 17 or claim 18 wherein the transgenic animal is a mammal, preferably a mouse or a sheep .