US20030074684A1 - Control of gene expression - Google Patents

Control of gene expression Download PDF

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Publication number
US20030074684A1
US20030074684A1 US09/997,905 US99790501A US2003074684A1 US 20030074684 A1 US20030074684 A1 US 20030074684A1 US 99790501 A US99790501 A US 99790501A US 2003074684 A1 US2003074684 A1 US 2003074684A1
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United States
Prior art keywords
plasmid
gene
pvy
sequence
cell
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US09/997,905
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English (en)
Inventor
Michael Graham
Robert Rice
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STATE OF QUEENSLAND THROUGH ITS Department OF PRIMARY INDUSTRIES
Benitec Australia Ltd
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Individual
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Family has litigation
First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=25645735&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=US20030074684(A1) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Priority claimed from AUPP2492A external-priority patent/AUPP249298A0/en
Priority claimed from AUPP2499A external-priority patent/AUPP249998A0/en
Application filed by Individual filed Critical Individual
Priority to US09/997,905 priority Critical patent/US20030074684A1/en
Assigned to BENITEC AUSTRALIA LTD., STATE OF QUEENSLAND THROUGH ITS DEPT. OF PRIMARY INDUSTRIES reassignment BENITEC AUSTRALIA LTD. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: GRAHAM, MICHAEL WAYNE, RICE, ROBERT NORMAN
Publication of US20030074684A1 publication Critical patent/US20030074684A1/en
Abandoned legal-status Critical Current

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    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron

Definitions

  • the present invention relates generally to a method of modifying gene expression and to synthetic genes for modifying endogenous gene expression in a call, tissue or organ of a transgenic organism, in particular a transgenic animal or plant. More particularly, the present invention utilises recombinant DNA technology to post-transcriptionally modify or modulate the expression of a target gene in a call, tissue, organ or whole organism, thereby producing novel phenotypes. Novel synthetic genes and genetic constructs which are capable of repressing delaying or otherwise reducing the expression of an endogenous gene or a target gene in an organism when introduced thereto are also provided.
  • derived from shall be taken to indicate that a specified integer may be obtained from a particular specified source or species, albeit not necessarily directly from that specified source or species.
  • Sequence identity numbers (SEQ ID NOS.) containing nucleotide and amino acid sequence information included in this specification are collected after the Abstract and have been prepared using the programme Patentln Version 2.0. .
  • Each nucleotide or amino acid sequence is identified in the sequence listing by the numeric indicator ⁇ 210>followed by the sequence identifier (e.g. ⁇ 210>1, ⁇ 210 >2, etc).
  • the length, type of sequence (DNA, protein (PRT), etc) and source organism for each nucleotide or amino acid sequence are indicated by information provided in the numeric indicator fields ⁇ 0211>, ⁇ 212>and ⁇ 213>, respectively.
  • Nucleotide and amino acid sequences referred to in the specification are defined by the information provided in numeric indicator field ⁇ 400>followed by the sequence identifier (eg. ⁇ 400>1, ⁇ 400>2, etc).
  • nucleotide residues referred to herein are those recommended by the IUPAC-IUB Biochemical Nomenclature Commission, wherein A represents Adenine, C represents Cytosine, G represents Guanine, T represents thymine, Y represents a pyrimidine residue, R represents a purine residue, M represents Adenine or Cytosine, K represents Guanine or Thymine, S represents Guanine or Cytosine, W represents Adenine or Thymine, H represents a nucleotide other than Guanine, B represents a nucleotide other than Adenine, V represents a nucleotide other than Thymine, D represents a nucleotide other than Cytosine and N represents any nucleotide residue.
  • Controlling metabolic pathways in eukaryotic organisms is desirable for the purposes of producing novel traits therein or introducing novel traits into a particular cell, tissue or organ of said organism. Whilst recombinant DNA technology has provided significant progress in an understanding of the mechanisms regulating eukaryotic gene expression, much less progress has been made in the actual manipulation of gene expression to produce novel traits. Moreover, there are only limited means by which human intervention may lead to a modulation of the level of eukaryotic gene expression.
  • RNA molecule which is transcribed from the complementary strand of a nuclear gene to that which is normally transcribed and capable of being translated into a polypeptide.
  • a double-stranded RNA may form by base pairing between the complementary nucleotide sequences, to produce a complex which is translated at low efficiency and/or degraded by intracellular ribonuclease enzymes prior to being translated.
  • an endogenous gene in a plant cell, tissue or organ may be suppressed when one or more copies of said gene, or one or more copies of a substantially similar gene are introduced into the cell.
  • this approach has been postulated to involve transcriptional repression, in which case somatically-heritable repressed states of chromatin are formed or alternatively, a post-transcriptional silencing wherein transcription occurs normally but the RNA products of the co-suppressed genes are subsequently eliminated.
  • the invention is based in part on the surprising discovery by the inventors that cells which exhibit one or more desired traits can be produced and selected from transformed cells comprising a nucleic acid molecule operably linked to a promoter, wherein the transcription product of the nucleic acid molecule comprises a nucleotide sequence which is substantially identical to the nucleotide sequence of a transcript of an endogenous or non-endogenous target gene, the expression of which is intended to be modulated.
  • the transformed cells are regenerated into whole tissues, organs or organisms capable of exhibiting novel traits, in particular virus resistance and modified expression of endogenous genes.
  • one aspect of the present invention provides a method of modulating the expression of a target gene in a plant cell, tissue or organ comprising (a) providing one or more dispersed or foreign nucleic acid molecules which include multiple copies of a nucleotide sequence, each of which is substantially identical to or complementary to the nucleotide sequence of the target gene or a region thereof, and (b) transfecting the plant cell, tissue or organ with the dispersed or foreign nucleic acid molecules for a time and under conditions sufficient for expression of at least two of the multiple copies.
  • the target gene may be a gene which is endogenous to the cell or alternatively, a foreign gene such as a viral or foreign genetic sequence, amongst others.
  • a foreign gene such as a viral or foreign genetic sequence, amongst others.
  • the target gene is a viral genetic sequence.
  • the invention is particularly useful in the modulation of eukaryotic gene expression, in particular the modulation of plant or animal gene expression and even more particularly in the modulation of expression of genes derived from crops, vertebrate and invertebrate animals, such as insects, aquatic animals (eg. fish, shellfish, molluscs, crustaceans such as crabs, lobsters and prawns, avian animals and mammals, amongst others).
  • genes derived from crops, vertebrate and invertebrate animals such as insects, aquatic animals (eg. fish, shellfish, molluscs, crustaceans such as crabs, lobsters and prawns, avian animals and mammals, amongst others).
  • a variety of traits are selectable with appropriate procedures and sufficient numbers of transformed cells. Such traits include, but are not limited to, visible traits, disease-resistance traits, and pathogen-resistance traits.
  • the modulatory effect is applicable to a variety of genes expressed in animals including, for example, endogenous genes responsible for cellular metabolism or cellular transformation, including oncogenes, transcription factors and other genes which encode polypeptides involved in cellular metabolism.
  • an alteration in the pigment production in mice can be engineered by targeting the expression of the tyrosinase gene therein.
  • This provides a novel phenotype of albinism in black mice.
  • a genetic construct which comprises multiple copies of nucleotide sequence encoding a viral replicase, polymerase, coat protein or uncoating gene, or protease protein, may be introduced into a cell where it is expressed, to confer immunity against the virus upon the cell.
  • the dispersed nucleic acid molecule or foreign nucleic acid molecule will generally comprise a nucleotide sequence having greater than about 85% identity to the target gene sequence, however, a higher homology might produce a more effective modulation of expression of the target gene sequence. Substantially greater homology, or more than about 90% is preferred, and even more preferably about 95% to absolute identity is desirable.
  • the introduced dispersed nucleic acid molecule or foreign nucleic acid molecule sequence needing less than absolute homology, also need not be full length, relative to either the primary transcription product or fully processed mRNA of the target gene. A higher homology in a shorter than full length sequence compensates for a longer less homologous sequence. Furthermore, the introduced sequence need not have the same intron or exon pattern, and homology of non-coding segments will be equally effective.
  • a second aspect of the present invention provides a method of modulating the expression of a target gene in a plant cell, tissue or organ, said method comprising:
  • a third aspect of the invention provides a method of conferring resistance or immunity to a viral pathogen upon a plant cell, tissue, organ or whole organism, comprising:
  • a fourth aspect of the present invention provides a genetic construct comprising multiple structural gene sequences, wherein each of said structural gene sequences is substantially identical to a target gene in a plant cell, and wherein said multiple structural gene sequences are placed operably under the control of a single promoter sequence which is operable in said cell, wherein at least one of said structural gene sequences is placed operably in the sense orientation under the control of said promoter sequence and at least one other of said structural gene sequences is placed operably in the antisense orientation under the control of said promoter sequence, and wherein at least one structural gene sequence that is placed in the sense orientation relative to said promoter and at least one structural gene sequence that is placed in the antisense orientation relative to said promoter are spaced from each other by a nucleic acid stuffer fragment.
  • a fifth aspect of the present invention provides a genetic construct which is capable of modulating the expression of a target gene in a plant cell, which is transfected with said construct, wherein said construct comprises multiple copies of a structural gene sequence, wherein each copy comprises a nucleotide sequence which is substantially identical to said target gene or a derivative of said target gene and wherein said multiple copies are placed operably under the control of a single promoter sequence which is operable in said cell, wherein at least two of said copies are placed operably in the sense orientation under the control of said promoter sequence.
  • a sixth aspect of the present invention provides a genetic construct which is capable of modulating the expression of a target gene in a plant cell, which is transfected with said construct, wherein said construct comprises multiple structural gene sequences wherein each of said structural gene sequences is separately placed under the control of a promoter which is operable in said cell, and wherein each of said structural gene sequences comprises a nucleotide sequence which is substantially identical to said target gene or a derivative of said target gene, wherein at least one of said structural gene sequences is placed operably in the sense orientation under the control of an individual promoter sequence.
  • FIG. 1 is a diagrammatic representation of the plasmid pEGFP-N1 MCS.
  • FIG. 2 is a diagrammatic representation of the plasmid pCMV.cass.
  • FIG. 3 is a diagrammatic representation of the plasmid pCMV.SV40L.cass.
  • FIG. 4 is a diagrammatic representation of the plasmid pCMV.SV40LR.cass.
  • FIG. 5 is a diagrammatic representation of the plasmid pCR.Bgl-GFP-Bam.
  • FIG. 6 is a diagrammatic representation of the plasmid pBSII(SK+).EGFP.
  • FIG. 7 is a diagrammatic representation of the plasmid pCMV.EGFP.
  • FIG. 8 is a diagrammatic representation of the plasmid pCR.SV40L.
  • FIG. 9 is a diagrammatic representation of the plasmid pCR.BEV.1.
  • FIG. 10 is a diagrammatic representation of the piasmid pCR.BEV.2.
  • FIG. 11 is a diagrammatic representation of the plasmid pCR.BEV.3.
  • FIG. 12 is a diagrammatic representation of the plasmid pCMV,EGFP.BEV2.
  • FIG. 13 is a diagrammatic representation of the plasmid pCMV.BEV.2.
  • FIG. 14 is a diagrammatic representation of the plasmid pCMV.BEV.3.
  • FIG. 15 is a diagrammatic representation of the plasmid pCMV.VEB.
  • FIG. 16 is a diagrammatic representation of the piasmid pCMV.BEV.GFP.
  • FIG. 17 is a diagrammatic representation of the plasmid pCMV.BEV.SV40L-0.
  • FIG. 18 is a diagrammatic representation of the plasmid pCMV.O.SV40L.BEV.
  • FIG. 19 is a diagrammatic representation of the plas mid pCMV.O.SV40L.VEB,
  • FIG. 20 is a diagrammatic representation of the plasmid pCMV.BEVX2.
  • FIG. 21 is a diagrammatic representation of the plasmid pCMV.BEVx3.
  • FIG. 22 is a diagrammatic representation of the plasmid pCMV.BEVx4.
  • FIG. 23 is a diagrammatic representation of the plasmid pCMV.BEV.SV40L.BEV.
  • FIG. 24 is a diagrammatic representation of the plasmid pCMV.BEV.SV40L.VEB.
  • FIG. 25 is a diagrammatic representation of the plasmid pCMV.BEV.GFP.VEB.
  • FIG. 26 is a diagrammatic representation of the plasmid pCMV.EGFP.BEV2.PFG.
  • FIG. 27 is a diagrammatic representation of the plasmid pCMV.BEV.SV40LR.
  • FIG. 28 is a diagrammatic representation of the plasmid pCDNA3.Galt.
  • FIG. 29 is a diagrammatic representation of the plasmid pCMV.Galt.
  • FIG. 30 is a diagrammatic representation of the plasmid pCMV.EGFP.Gait.
  • FIG. 31 is a diagrammatic representation of the plasmid pCMV.GaIt.GFP.
  • FIG. 32 is a diagrammatic representation of the plasmid pCMV.Galt.SV40L.0,
  • FIG. 33 is a diagrammatic representation of the plasmid pCMV.Gait.SV40L.tiaG.
  • FIG. 34 is a diagrammatic representation of the plasmid pCMV.O.SV40L.Galt.
  • FIG. 35 is a diagrammatic representation of the plasmid pCMV.Gaitx2.
  • FIG. 36 is a diagrammatic representation of the plasmid pCMV.Galtx4.
  • FIG. 37 is a diagrammatic representation of the plasmid pCMV.Galt.SV40L.Gait.
  • FIG. 38 is a diagrammatic representation of the plasmid pCMV.Galt.SV40L.tIaG.
  • FIG. 39 is a diagrammatic representation of the plasmid pCMV.Galt.GFP.tlaG.
  • FIG. 40 is a diagrammatic representation of the plasmid pCMV.EGFP.GaIt.PFG.
  • FIG. 41 is a diagrammatic representation of the plasmid pCMV.Galt.SV40LR.
  • FIG. 42 is a diagrammatic representation of the plasmid pART7.
  • FIG. 43 is a diagrammatic representation of the plasmid pART7.35S.SCBV.cass.
  • FIG. 44 Is a diagrammatic representation of the p.asmid pBCPVY.
  • FIG. 45 is a diagrammatic representation of the pfasmid pSP72.PVY.
  • FIG. 46 is a diagrammatic representation of the plasmid pCIapBC.PVY.
  • FIG. 47 is a diagrammatic representation of the plasmid pBC.PVYx2.
  • FIG. 48 is a diagrammatic representation of the piasmid pSP72.PVYx2.
  • FIG. 49 is a diagrammatic representation of the plasmid pBC.PVYx3.
  • FIG. 50 is a diagrammatic representation of the plasmid pBC.PVYx4.
  • FIG. 51 is a diagrammatic representation of the plasmid pBC.PVY.LNYV.
  • FIG. 52 is a diagrammatic representation of the plasmid pBC.PVY.LNYV.PVY.
  • FIG. 53 is a diagrammatic representation of the plasmid pBC.PVY.LNYV.YVP.
  • FIG. 54 is a diagrammatic representation of the plasmid pBC.PVY.LNYV.YVP.
  • FIG. 55 is a diagrammatic representation of the plasmid pART27.PVY
  • FIG. 56 is a diagrammatic representation of the plasmid pART27.35S.PVY.SCBV.O.
  • FIG. 57 is a diagrammatic representation of the plasmid pART27.35S.O.SCBV.PVY.
  • FIG. 58 is a diagrammatic representation of the plasmid pART27.35S.O.SCBV.YVP.
  • FIG. 59 is a diagrammatic representation of the plasmid pART7.PVYx2.
  • FIG. 60 is a diagrammatic representation of the plasmid pART7.PVYx3.
  • FIG. 61 is a diagrammatic representation of the plasmid pART7.PVYx4.
  • FIG. 62 is a diagrammatic representation of the plasmid pART7.PVY.LNYV.PVY.
  • FIG. 63 is a diagrammatic representation of the plasmid pART7.PVY.LNYV.YVP.
  • FIG. 64 is a diagrammatic representation of the piasmid pART7. PVY.LNYV.YVP.
  • FIG. 65 is a diagrammatic representation of pART7.35S.PVY.SCBV.YVP.
  • FIG. 66 is a diagrammatic representation of pART7.35S.PVYx3.SCBV.YVPx3.
  • FIG. 67 is a diagrammatic representation of pART7.PVYx3.LNYV.YVPx3.
  • FIG. 68 is a diagrammatic representation of the plasmid pART7,PVYMULTI.
  • FIG. 70 shows an example of Southern blot analysis of transgenic porcine kidney cells (PK) which had been transformed with the construct pCMV.EGFP.
  • Genomic DNA was isolated from PK-1 cells and transformed lines, digested with the restriction endonuclease BamHl and probed with a 32 P-dCTP labeled EGFP DNA fragment.
  • Lane A is a molecular weight marker where sizes of each fragment are indicated in kilobases (kb);
  • Lane B is the parental cell line PK-1.
  • Lane C is A4, a transgenic EGFP-expressing PK-1 cell line;
  • Lane D is C9, a transgenic non-expressing PK-1 cell line.
  • FIG. 71 shows micrographs of CRIB-1 cells and a CRIB-1 transformed line [CRIB-1 BGI2 # 19(tol)] prior to and 48 hrs after infection with identical titres of BEV.
  • A CRIB-1 cells prior to BEV infection
  • B CRIB-1 cells 48 hrs after BEV infection
  • C CRIB-1 BGI2 # 19(tol) cells prior to infection with BEV
  • D CRIB-1 BGI2 # 19(tol) 48 hrs after BEV infection.
  • FIG. 72 shows levels of pigmentation in B16 cells and B16 cells transformed with pCMV.TYR.BGI2.RYT.
  • Cell lines are, from left to right: B16, B16 2.1.6, B16 2.1.11, B16 3.1.4, B16 3.1.15, B16 4.12.2 and B16 4.12.3.
  • B16, B16 2.1.6, B16 2.1.11, B16 3.1.4, B16 3.1.15, B16 4.12.2 and B16 4.12.3 For further details refer to Example 9.
  • FIG. 73 shows immunofluorescent micrographs of MDA-MB-468 cells and MDA-MB-468 cells transformed with pCMV.HER2.BGI2.2REH stained for HER-2.
  • B MDA-MB-468 cells stained with only the secondary antibody
  • C MDA-MB-468 1.4 cells stained for HER-2
  • D MDA-MB-468 1.10 cells stained for HER-2.
  • FIG. 74 shows FACS analyses of HER-2 expression in (A) MDA-MB-468 cells; (B) MDA-MB-468 1.4 cells; (C) MDA-MB-468 1.10 cells.
  • A MDA-MB-468 cells
  • B MDA-MB-468 1.4 cells
  • C MDA-MB-468 1.10 cells.
  • FIG. 75 is a histograph showing viable cell counts after transfection with YB-1-related gene constructs and oligonucleotides. Viable cells were counted in quadruplicate samples with a haemocytometer following staining with trypan blue. Column heights show the average cell count of two independent transfection experiments and vertical bars indicate the standard deviation.
  • FIG. 76 A Northern blot showing levels of GFP expression in MM96L cells and MM96L lines transformed with pCMV.EGFP. 10 ⁇ g of total RNA from the indicated cell lines were electrophoresed on agarose gels and transferred to a nylon membrane. The filter was probed with a radio-labelled fragment derived from the EGFP gene. B Photograph showing ethidium bromide-stained ribosomal RNAs from the RNA samples probed in A; the equal intensities indicated similar amounts of RNA from each cell line were probed.
  • FIG. 77 Real-Time RT-PCR analysis of transformed cell lines for EGFP mRNA levels and EGFP RNA transcribed from the EGFP transgene in nuclear run-on assays.
  • C Glyceraldehyde phosphate dehydrogenase (GAPD) mRNA levels in MM96L lines 3, 9 and 18(as in FIG. 76).
  • FIG. 78 Relative mRNA levels and RNA transcribed from the EGFP transgene in nuclear run-on assays, from the data shown in FIG. 77.
  • the EGFP gene in line 9 is transcribed but EGFP mRNA levels are extremely low, signifying post-transcriptional gene silencing (co-suppression).
  • the present invention provides a method of modulating the expression of a target gene in a cell, tissue or organ, said method at least comprising the step of introducing to said cell, tissue or organ one or more dispersed nucleic acid molecules or foreign nucleic acid molecules comprising multiple copies of a nucleotide sequence which is substantially identical to the nucleotide sequence of said target gene or a region thereof or complementary thereto for a time and under conditions sufficient for translation of the mRNA product of said target gene to be modified, subject to the proviso that the transcription of said mRNA product is not exclusively repressed or reduced.
  • multiple copies is meant that two or more substantially identical (as defined below) copies of a nucleotide sequence are present in the same or different orientation, on the same nucleic acid molecule.
  • direct repeat is used in contradistinction to the term “inverted repeat” such that a direct repeat is a 5′-3′.5′-3′repeat (with or without other nucleotides between the repeated sequences).
  • An inverted repeat is a 5′-3′,3′ ⁇ 5′sequence (the 3′ ⁇ 5′sequence may also be called a “reverse complement” or antisense of the 5′ ⁇ 3′sequence) such that the transcription product of the inverted repeat (e.g. mRNA) may form a hairpin RNA structure.
  • the term “tandem” is used by those skilled in the art to indicate repeats separated by no or relatively few (relative to the length of the repeated sequence) intervening nucleotides.
  • Repeats may optionally be separated by a stuffer fragment or intergenic region to facilitate secondary structure formation between each repeat where this is required.
  • the stuffer fragment may comprise any combination of nucleotide or amino acid residues, carbohydrate molecules or oligosaccharide molecules or carbon atoms or a homologue, analogue or derivative thereof which is capable of being linked covalently to a nucleic acid molecule.
  • the stuffer fragment comprises a sequence of nucleotides or a homologue, analogue or derivative thereof.
  • the stuffer fragment may serve as an intron sequence placed between the 3′-splice site of the structural gene nearer the 5′-end of the gene and the 5′-splice site of the next downstream unit thereof.
  • the stuffer fragment placed there between should not include an in-frame translation stop codon, absent intron/exon splice junction sequences at both ends of the stuffer fragment or the addition of a translation start codon at the 5′end of each unit, as will be obvious to those skilled in the art.
  • Stuffer fragments can include those which encode detectable marker proteins or biologically-active analogues and derivatives thereof, for example luciferase, ⁇ -galacturonase, ⁇ -galactosidase, chloramphenicol acetyltransferase or green fluorescent protein, amongst others. Additional stuffer fragments are not excluded.
  • the detectable marker or an analogue or derivative thereof serves to indicate the expression of the synthetic gene of the invention in a cell, tissue or organ by virtue of its ability to confer a specific detectable phenotype thereon, preferably a visually-detectable phenotype.
  • the term “modulating” shall be taken to mean that expression of the target gene is reduced in amplitude and/or the timing of gene expression is delayed and/or the developmental or tissue-specific or cell-specific pattern of target gene expression is altered, compared to the expression of said gene in the absence of the inventive method described herein.
  • the present invention is directed to a modulation of gene expression which comprises the repression, delay or reduction in amplitude of target gene expression in a specified cell, tissue or organ of a eukaryotic organism, in particular a plant such as a monocotyledonous or dicotyledonous plant, or a human or other animal and even more particularly a vertebrate and invertebrate animal, such as an insect, aquatic animal (eg. fish, shellfish, mollusc, crustacean such as a crab, lobster or prawn, an avian animal or a mammal, amongst others).
  • a modulation of gene expression which comprises the repression, delay or reduction in amplitude of target gene expression in a specified cell, tissue or organ of a eukaryotic organism, in particular a plant such as a monocotyledonous or dicotyledonous plant, or a human or other animal and even more particularly a vertebrate and invertebrate animal, such as an insect, aquatic animal (
  • target gene expression is completely inactivated by the dispersed nucleic acid molecules or foreign nucleic acid molecules which has been introduced to the cell, tissue or organ.
  • the reduced or eliminated expression of the target gene which results from the performance of the invention may be attributed to reduced or delayed translation of the RNA transcription product of the target gene or alternatively, the prevention of translation of said RNA, as a consequence of sequence-specific degradation of the RNA transcript of the target gene by an endogenous host cell system.
  • RNA transcript of the target gene sequence-specific degradation of the RNA transcript of the target gene occurs either prior to the time or stage when the RNA transcript of the target gene would normally be translated or alternatively, at the same time as the RNA transcript of the target gene would normally be translated. Accordingly, the selection of an appropriate promoter sequence to regulate expression of the introduced dispersed nucleic acid molecule or foreign nucleic acid molecule is an important consideration to optimum performance of the invention. For this reason, strong constitutive promoters or inducible promoter systems are especially preferred for use in regulating expression of the introduced dispersed nucleic acid molecules or foreign nucleic acid molecules.
  • the present invention clearly encompasses reduced expression wherein reduced expression of the target gene is effected by lowered transcription, subject to the proviso that a reduction in transcription is not the sole mechanism by which this occurs and said reduction in transcription is at least accompanied by reduced translation of the steady-state mRNA pool of the target gene.
  • the target gene may be a genetic sequence which is endogenous to the cell or alternatively, a non-endogenous genetic sequence, such as a genetic sequence which is derived from a virus or other foreign pathogenic organism and is capable of entering a cell and using the cell's machinery following infection.
  • the target gene is a non-endogenous genetic sequence to the cell
  • the present invention is particularly useful in the prophylactic and therapeutic treatment of viral infection of an animal cell or for conferring or stimulating resistance against said pathogen.
  • the target gene comprises one or more nucleotide sequences of a viral pathogen of a plant or an animal cell, tissue or organ.
  • the viral pathogen may be a retrovirus, for example a lentivirus such as the immunodeficiency viruses, a single-stranded (+) RNA virus such as bovine enterovirus (BEV) or Sinbis alphavirus.
  • a retrovirus for example a lentivirus such as the immunodeficiency viruses, a single-stranded (+) RNA virus such as bovine enterovirus (BEV) or Sinbis alphavirus.
  • the target gene can comprise one or more nucleotide sequences of a viral pathogen of an animal cell, tissue or organ, such as but not limited to a double-stranded DNA virus such as bovine herpes virus or herpes simplex virus I (HSV II), amongst others.
  • a double-stranded DNA virus such as bovine herpes virus or herpes simplex virus I (HSV II), amongst others.
  • the virus pathogen is preferably a potyvirus, caulimovirus, badnavirus, geminivirus, reovirus, rhabdovirus, bunyavirus, tospovirus, tenuivirus, tombusvirus, luteovirus, sobemovirus, bromovirus, cucomovirus, ilavirus, alfamovirus, tobamovirus, tobravirus, potexvirus and clostrovirus, such as but not limited to CaMV, SCSV, PVX, PVY, PLRV, and TMV, amongst others.
  • virus-encoded functions may be complemented in trans by polypeptides encoded by the host cell.
  • the replication of the bovine herpes virus genome in the host cell may be facilitated by host cell DNA polymerases which are capable of complementing an inactivated viral DNA polymerase gene.
  • a further alternative embodiment of the invention provides for the target gene to encode a viral or foreign polypeptide which is not capable of being complemented by a host cell function, such as a virus-specific genetic sequence.
  • exemplary target genes according to this embodiment of the invention include, but are not limited to genes which encode virus coat proteins, uncoating proteins and RNA-dependent DNA polymerases and RNA-dependent RNA polymerases, amongst others.
  • the target gene is the BEV RNA-dependent RNA polymerase gene or a homologue, analogue or derivative thereof or PVY Nia protease-encoding sequences.
  • the cell in which expression of the target gene is modified may be any cell which is derived from a multicellular animal, including cell and tissue cultures thereof.
  • the animal cell is derived from an anthropod, nematode, reptile, amphibian, bird, human or other mammal.
  • Exemplary animal cells include embryonic stem cells, cultured skin fibroblasts, neuronal cells, somatic cells, haematopoietic stem cells, T-cells and immortalised cell lines such as COS, VERO, HeLa, mouse C127, Chinese hamster ovary (CHO), WI-38, baby hamster kidney (BHK) or MDBK cell lines, amongst others.
  • Such cells and cell lines are readily available to those skilled in the art.
  • the tissue or organ in which expression of the target gene is modified may be any tissue or organ comprising such animal cells.
  • the plant cell is derived from a monocotyledonous or dicotyledonous plant species or a cell line derived therefrom.
  • the term “dispersed nucleic acid molecule” shall be taken to refer to a nucleic acid molecule which comprises one or more multiple copies of a nucleotide sequence which is substantially identical or complementary to the nucleotide sequence of a gene which originates from the cell, tissue or organ into which said nucleic acid molecule is introduced, wherein said nucleic acid molecule is non-endogenous in the sense that it is introduced to the cell, tissue or organ of an animal via recombinant means and will generally be present as extrachromosomal nucleic acid or alternatively, as integrated chromosomal nucleic acid which is genetically-unlinked to said gene.
  • the “dispersed nucleic acid molecule” will comprise chromosomal or extrachromosomal nucleic acid which is unlinked to the target gene against which it is directed in a physical map, by virtue of their not being tandemly-linked or alternatively, occupying a different chromosomal position on the same chromosome or being localised on a different chromosome or present in the cell as an episome, plasmid, cosmid or virus particle.
  • foreign nucleic acid molecule an isolated nucleic acid molecule which has one or more multiple copies of a rucleotide sequence which originates from the genetic sequence of an organism which is different from the organism to which the foreign nucleic acid molecule is introduced. This definition encompasses a nucleic acid molecule which originates from a different individual of the same lowest taxonomic grouping (i.e.
  • nucleic acid molecule which originates from a different individual of a different taxonomic grouping as the taxonomic grouping to which said nucleic acid molecule is introduced, such as a gene derived from a viral pathogen.
  • a target gene against which a foreign nucleic acid molecule acts in the performance of the invention may be a nucleic acid molecule which has been introduced from one organism to another organism using transformation or introgression technologies.
  • Exemplary target genes according to this embodiment of the invention include the green fluorescent protein-encoding gene derived from the jellyfish Aequoria Victoria (Prasher et aL.,1992; International Patent Publication No.
  • tyrosinase genes and in particular the murine tyrosinase gene (Kwon et aL,1988), the Escherichia coli tacd gene which is capable of encoding a polypeptide repressor of the lacZ gene, the porcine ⁇ 1,3-galactosyltransferase gene (NCBI Accession No. L36535) exemplified herein, and the PVY and BEV structural genes exemplified herein or a homologue, analogue or derivative of said genes or a complementary nucleotide sequence thereto.
  • the present invention is further useful for simultaneously targeting the expression of several target genes which are co-expressed in a particular cell, for example by using a dispersed nucleic acid molecule or foreign nucleic acid molecule which comprises nucleotide sequences which are substantially identical to each of said co-expressed target genes.
  • substantially identical is meant that the introduced dispersed or foreign nucleic acid molecule of the invention and the target gene sequence are sufficiently identical at the nucleotide sequence level to permit base-pairing there between under standard intracellular conditions.
  • the nucleotide sequence of each repeat in the dispersed or foreign nucleic acid molecule of the invention and the nucleotide sequence of a part of the target gene sequence are at least about 80-85% identical at the nucleotide sequence level, more preferably at least about 85-90% identical, even more preferably at least about 90-95% identical and still even more preferably at least about 95-99% or 100% identical at the nucleotide sequence level.
  • the present invention is not limited by the precise number of repeated sequences in the dispersed nucleic acid molecule or the foreign nucleic acid molecule of the invention, it is to be understood that the present invention requires at least two copies of the dispersed foreign nucleic acid molecule of the target gene sequence to be expressed in the cell.
  • the multiple copies of the target gene sequence are presented in the dispersed nucleic acid molecule or the foreign nucleic acid molecule as tandem inverted repeat sequences and/or tandem direct repeat sequences.
  • Such configurations are exemplified by the “test plasmids” described herein that comprise Galt, BEV or PVY gene regions.
  • the dispersed or foreign nucleic acid molecule which is introduced to the cell, tissue or organ comprises RNA or DNA.
  • the dispersed or foreign nucleic acid molecule further comprises a nucleotide sequence or is complementary to a nucleotide sequence which is capable of encoding an amino acid sequence encoded by the target gene.
  • Standard methods may be used to introduce the dispersed nucleic acid molecule or foreign nucleic acid molecule into the cell, tissue or organ for the purposes of modulating the expression of the target gene.
  • the nucleic acid molecule may be introduced as naked DNA or RNA, optionally encapsulated in a liposome, in a virus particle as attenuated virus or associated with a virus coat or a transport protein or inert carrier such as gold or as a recombinant viral vector or bacterial vector or as a genetic construct, amongst others.
  • Administration means include injection and oral ingestion (e.g. in medicated food material), amongst others.
  • the subject nucleic acid molecules may also be delivered by a live delivery system such as using a bacterial expression system optimised for their expression in bacteria which can be incorporated into gut flora.
  • a viral expression system can be employed.
  • one form of viral expression is the administration of a live vector generally by spray, feed or water where an infecting effective amount of the live vector (e.g. virus or bacterium) is provided to an animal.
  • Another form of viral expression system is a non-replicating virus vector which is capable of infecting a cell but not replicating therein.
  • the non-replicating viral vector provides a means of introducing to the human or animal subject genetic material for transient expression therein.
  • the mode of administering such a vector is the same as a live viral vector.
  • the carriers, excipients and/or diluents utilised in delivering the subject nucleic acid molecules to a host cell should be acceptable for human or veterinary applications.
  • Such carriers, excipients and/or diluents are well-known to those skilled in the art.
  • Carriers and/or diluents suitable for veterinary use include any and all solvents, dispersion media, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the composition is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • the invention provides a method of modulating the Expression of a target gene in a cell, tissue or organ, said method at least comprising the steps of:
  • nucleotide sequences for targeting expression of the target gene may be employed.
  • multiple copies of specific regions of characterised genes may be cloned in operable connection with a suitable promoter and assayed for efficacy in reducing target gene expression.
  • shotgun libraries comprising multiple copies of genetic sequences may be produced and assayed for their efficacy in reducing target gene expression.
  • the advantage associated with the latter approach is that it is not necessary to have any prior knowledge of the significance of any particular target gene in specifying. an undesirable phenotype in the cell.
  • shotgun libraries comprising virus sub-genomic fragments may be employed and tested directly for their ability to confer virus immunity on the animal host cell, without prior knowledge of the role which any virus genes play in pathogenesis of the host cell.
  • shotgun library is a set of diverse nucleotide sequences wherein each member of said set is preferably contained within a suitable plasmid, cosmid, bacteriophage or virus vector molecule which is suitable for maintenance and/or replication in a cellular host.
  • shotgun library includes a representative library, in which the extent of diversity between the nucleotide sequences is numerous such that all sequences in the genome of the organism from which said nucleotide sequences is derived are present in the “set” or alternatively, a limited library in which there is a lesser degree of diversity between said sequences.
  • shotgun library further encompasses random nucleotide sequences, wherein the nucleotide sequence comprises viral or cellular genome fragments, amongst others obtained for example by shearing or partial digestion of genomic DNA using restriction endonucleases, amongst other approaches.
  • a “shotgun library” further includes cells, virus particles and bacteriophage particles comprising the individual nucleotide sequences of the diverse set.
  • Preferred shotgun libraries according to this embodiment of the invention are “representative libraries”, comprising a set of tandem repeated nucleotide sequences derived from a viral pathogen of a plant or an animal.
  • the shotgun library comprises cells, virus particles or bacteriophage particles comprising a diverse set of tandem-repeated nucleotide sequences which encode a diverse set of amino acid sequences, wherein the members of said diverse set of nucleotide sequences are placed operably under the control of a promoter sequence which is capable of directing the expression of said tandem-repeated nucleotide sequence in the cell.
  • nucleotide sequence of each unit in the tandem-repeated sequence may comprise at least about 20 to 200 nucleotides in length.
  • the introduced nucleic acid molecule is preferably in an expressible form.
  • “expressible form” is meant that the subject nucleic acid molecule is presented in an arrangement such that it may be expressed in the cell, tissue, organ or whole organism, at least at the transcriptional level (i.e. it is expressed in the animal cell to yield at least an RNA product which is optionally translatable or translated to produce a recombinant peptide, oligopeptide or polypeptide molecule).
  • a synthetic gene or a genetic construct comprising said synthetic gene is produced, wherein said synthetic gene comprises a nucleotide sequence as described supra in operable connection with a promoter sequence which is capable of regulating expression therein.
  • the subject nucleic acid molecule will be operably connected to one or more regulatory elements sufficient for eukaryotic transcription to occur.
  • a classical genomic gene consisting of transcriptional and/or translational regulatory sequences and/or a coding region and/or non-translated sequences (i.e. introns, 5′- and 3′-untranslated sequences, or DNA and RNA viruses); and/or
  • a structural region corresponding to the coding regions optionally further comprising untranslated sequences and/or a heterologous promoter sequence which consists of transcriptional and/or translational regulatory regions capable of conferring expression characteristics on said structural region.
  • “gene” includes a nucleotide sequence coding for RNA other than mRNA.
  • gene is also used to describe synthetic or fusion molecules encoding all or part of a functional product, in particular a sense or antisense RNA product or a peptide, oligopeptide or polypeptide or a biologically-active protein.
  • synthetic gene refers to a non-naturally occurring gene as hereinbefore defined which preferably comprises at least one or more transcriptional and/or translational regulatory sequences operably linked to a structural gene sequence.
  • structural gene shall be taken to refer to a nucleotide sequence which is capable of being transcribed to produce RNA and optionally, encodes a peptide, oligopeptide, polypeptide or biologically active protein molecule.
  • RNA is capable of being translated into a peptide, oligopeptide, polypeptide or protein, for example if the RNA lacks a functional translation start signal or alternatively, if the RNA is antisense RNA
  • the present invention clearly encompasses synthetic genes comprising nucleotide sequences which are not capable of encoding peptides, oligopeptides, polypeptides or biologically-active proteins.
  • the present inventors have found that such synthetic genes may be advantageous in modifying target gene expression in cells, tissues or organs of a prokaryotic or eukaryotic organism.
  • structural gene region refers to that part of a synthetic gene which comprises a dispersed nucleic acid molecule or foreign nucleic acid molecule as described herein which is expressed in a cell, tissue or organ under the control of a promoter sequence to which it is operably connected.
  • a structural gene region may comprise one or more dispersed nucleic acid molecules and/or foreign nucleic acid molecules operably under the control of a single promoter sequence or multiple promoter sequences.
  • the structural gene region of a synthetic gene may comprise a nucleotide sequence which is capable of encoding an amino acid sequence or is complementary thereto.
  • a structural gene region which is used in the performance of the instant invention may also comprise a nucleotide sequence which encodes an animo acid sequence yet lacks a functional translation initiation codon and/or a functional translation stop codon and, as a consequence, does not comprise a complete open reading frame.
  • the term “structural gene region” also extends to a non-coding nucleotide sequences, such as 5′-upstream or 3′-downstream sequences of a gene which would not normally be translated in a eukaryotic cell which expresses said gene.
  • a structural gene region may also comprise a fusion between two or more open reading frames of the same or different genes.
  • the invention may be used to modulate the expression of one gene, by targeting different non-contiguous regions thereof or alternatively, to simultaneously modulate the expression of several different genes, including different genes of a multigene family.
  • the fusion may provide the added advantage of conferring simultaneous immunity or protection against several pathogens, by targeting the expression of genes in said several pathogens.
  • the fusion may provide more effective immunity against any pathogen by targeting the expression of more than one gene of that pathogen.
  • the optimum number of structural gene sequences to be included in the synthetic genes of the present invention may be determined empirically by those skilled in the art, without any undue experimentation and by following standard procedures such as the construction of the synthetic gene of the invention using recombinase-deficient cell lines, reducing the number of repeated sequences to a level which eliminates or minimises recombination events and by keeping the total length of the multiple structural gene sequence to an acceptable limit, preferably no more than 5-10kb, more preferably no more than 2-5kb and even more preferably no more than 0.5-2.0kb in length.
  • a stuffer fragment can be inserted between copies forming the palindrome.
  • the structural gene region comprises more than one dispersed nucleic acid molecule or foreign nucleic acid molecule it shall be referred to herein as a “multiple structural gene region” or similar term.
  • the present invention clearly extends to the use of multiple structural gene regions which preferably comprise a direct repeat sequence, inverted repeat sequence or interrupted palindrome sequence of a particular structural gene, dispersed nucleic acid molecule or foreign nucleic acid molecule, or a fragment thereof.
  • Each dispersed or foreign nucleic acid molecule contained within the multiple structural gene unit of the subject synthetic gene may comprise a nucleotide sequence which is substantially identical to a different target gene in the same organism.
  • Such an arrangement may be of particular utility when the synthetic gene is intended to provide protection against a pathogen in a cell, tissue or organ, in particular a viral pathogen, by modifying the expression of viral target genes.
  • the multiple structural gene may comprise nucleotide sequences (i.e. two or more dispersed or foreign nucleic acid molecules) which are substantially identical to two or more target genes selected from the list comprising DNA polymerase, RNA polymerase, Nia protease, and coat protein or other target gene which is essential for viral infectivity, replication or reproduction.
  • the structural gene units are selected such that the target genes to which they are substantially identical are normally expressed at approximately the same time (or later) in an infected call, tissue or organ as (than) the multiple structural gene of the subject synthetic gene is expressed under control of the promoter sequence.
  • the promoter controlling expression of the multiple structural gene will usually be selected to confer expression in the cell, tissue or organ over the entire life cycle of the virus when the viral target genes are expressed at different stages of infection.
  • the individual units of the multiple structural gene may be spatially connected in any orientation relative to each other, for example head-to-head, head-to-tail or tail-to-tail and all such configurations are within the scope of the invention.
  • the synthetic gene For expression in eukaryotic cells, the synthetic gene generally comprises, in addition to the nucleic acid molecule of the invention, a promoter and optionally other regulatory sequences designed to facilitate expression of the dispersed nucleic acid molecule or foreign nucleic acid molecule.
  • promoter includes the transcriptional regulatory sequences of a classical genomic gene, including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence and additional regulatory elements (i.e. upstream activating sequences, enhancers and silencers) which alter gene expression in response to developmental and/or external stimuli, or in a tissue-specific manner.
  • a promoter is usually, but not necessarily, positioned upstream or 5′, of a structural gene region, the expression of which it regulates.
  • the regulatory elements comprising a promoter are usually positioned within 2 kb of the start site of transcription of the gene.
  • promoter is also used to describe a synthetic or fusion molecule, or derivative which confers, activates or enhances expression of a nucleic acid molecule in a cell.
  • Preferred promoters may contain additional copies of one or more specific regulatory elements, to further enhance expression of the sense molecule and/or to alter the spatial expression and/or temporal expression of said sense molecule.
  • regulatory elements which confer copper inducibility may be placed adjacent to a heterologous promoter sequence driving expression of a sense molecule, thereby conferring copper ion inducibility on the expression of said molecule.
  • Placing a dispersed or foreign nucleic acid molecule under the regulatory control of a promoter sequence means positioning the said molecule such that expression is controlled by the promoter sequence. Promoters are generally positioned 5′(upstream) to the genes that they control. In the construction of heterologous promoter/structural gene combinations it is generally preferred to position the promoter at a distance from the gene transcription start site that is approximately the same as the distance between that promoter and the gene it controls in its natural setting, i.e., the gene from which the promoter is derived, As is known in the art, some variation in this distance can be accommodated without loss of promoter function.
  • the preferred positioning of a regulatory sequence element with respect to a heterologous gene to be placed under its control is defined by the positioning of the element in its natural setting, i.e., the genes from which it is derived. Again, as is known in the art, some variation in this distance can also occur.
  • promoters suitable for use in the synthetic genes of the present invention include viral, fungal, bacterial, animal and plant derived promoters capable of functioning in plant, animal, insect, fungal, yeast, or bacterial cells.
  • the promoter may regulate the expression of the structural gene component constitutively, or differentially with respect to the cell, the tissue or organ in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, or pathogens, or metal ions, or antibiotics, amongst others.
  • the promoter is capable of regulating expression of a nucleic acid molecule in a eukaryotic cell, tissue or organ, at least during the period of time over which the target gene is expressed therein and more preferably also immediately preceding the commencement of detectable expression of the target gene in said cell, tissue or organ.
  • promoters are particularly preferred for the purposes of the present invention or promoters which may be induced by virus infection or the commencement of target gene expression.
  • Plant-operable and animal-operable promoters are particularly preferred for use in the synthetic genes of the present invention.
  • preferred promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, CaMV 35S promoter, SCSV promoter, SCBV promoter and the like.
  • the promoter sequence is a constitutive strong promoter such as the CMV-IE promoter or the SV40 early promoter sequence, the SV40 late promoter sequence, the CaMV 35S promoter, or the SCBV promoter, amongst others.
  • a constitutive strong promoter such as the CMV-IE promoter or the SV40 early promoter sequence, the SV40 late promoter sequence, the CaMV 35S promoter, or the SCBV promoter, amongst others.
  • the terms “in operable connection with” or “operably under the control” or similar shall be taken to indicate that expression of the structural gene region or multiple structural gene region is under the control of the promoter sequence with which it is spatially connected; in a cell, tissue, organ or whole organism.
  • a structural gene region i.e. dispersed nucleic acid molecule or foreign nucleic acid molecule
  • multiple structural gene region is placed operably in connection with a promoter orientation relative to the promoter sequence, such that when it is transcribed an RNA product is synthesized which is capable of encoding a polypeptide product of the target gene or a fragment thereof.
  • the present invention is not to be limited to the use of such an arrangement and the invention clearly extends to the use of synthetic genes and genetic constructs wherein the a structural gene region or multiple structural gene region is placed in the “antisense” orientation relative to the promoter sequence, such that at least a part of the RNA transcription product thereof is complementary to the RNA encoded by the target gene or a fragment thereof.
  • dispersed nucleic acid molecule foreign nucleic acid molecule or multiple structural gene region comprises tandem direct and/or inverted repeat sequences of the target gene, all combinations of the above-mentioned configurations are encompassed by the invention.
  • the structural gene region or multiple structural gene region is operably connected to both a first promoter sequence and a second promoter sequence, wherein said promoters are located at the distal and proximal ends thereof such that at least one unit of said structural gene region or multiple structural gene region is placed in the “sense” orientation relative to the first promoter sequence and in the “antisense” orientation relative to the second promoter sequence.
  • the first and second promoters be different, to prevent competition there between for cellular transcription factors which bind thereto. The advantage of this arrangement is that the effects of transcription from the first and second promoters in reducing target gene expression in the cell may be compared to determine the optimum orientation for each nucleotide sequence tested.
  • the synthetic gene preferably contains additional regulatory elements for efficient transcription, for example a transcription termination sequence.
  • Terminator refers to a DNA sequence at the end of a transcriptional unit which signals termination of transcription. Terminators are 3′-non-translated DNA sequences which may contain a polyadenylation signal, which facilitates the addition of polyadenylate sequences to the 3′-end of a primary transcript. Terminators active in plant cells are known and described in the literature. They may be isolated from bacteria, fungi, viruses, animals and/or plants or synthesized de novo.
  • the terminator may be any terminator sequence which is operable in the cells, tissues or organs in which it is intended to be used.
  • terminators particularly suitable for use in the synthetic genes of the present invention include the SV40 polyadenylation signal, the HSV TK polyadenylation signal, the CYCl terminator, ADH terminator, SPA terminator, nopaline synthase (NOS) gene terminator of Agrobacterium tumefaciens, the terminator of the Cauliflower mosaic virus (CaMV) 35S gene, the zein gene terminator from Zea mays, the Rubisco small subunit gene (SSU) gene terminator sequences, subclover stunt virus (SCSV) gene sequence terminators, any rho-independent E coli terminator, or the lacZ alpha terminator, amongst others.
  • SSU Rubisco small subunit gene
  • SCSV subclover stunt virus
  • the terminator is the SV40 polyadenylation signal or the HSV TK polyadenylation signal which are operable in animal cells, tissues and organs, octopine synthase (OCS) or nopaline synthase (NOS) terminator active in plant cells, tissues or organs, or the lacZ alpha terminator which is active in prokaryotic cells.
  • OCS octopine synthase
  • NOS nopaline synthase
  • a genetic construct which comprises two or more structural gene regions or multiple structural gene regions wherein each of said structural gene regions is placed operably under the control of its own promoter sequence.
  • the orientation of each structural gene region may be varied to maximise its modulatory effect on target gene expression.
  • the promoters controlling expression of the structural gene unit are preferably different promoter sequences, to reduce competition there between for cellular transcription factors and RNA polymerases.
  • Preferred promoters are selected from those referred to supra.
  • the synthetic genes described supra are capable of being modified further, for example by the inclusion of marker nucleotide sequences encoding a detectable marker enzyme or a functional analogue or derivative thereof, to facilitate detection of the synthetic gene in a cell, tissue or organ in which it is expressed.
  • the marker nucleotide sequences will be present in a translatable format and expressed, for example as a fusion polypeptide with the translation product(s) of any one or more of the structural genes or alternatively as a non-fusion polypeptide.
  • the synthetic genes of the present invention may be introduced to a suitable cell, tissue or organ without modification as linear DNA in the form of a genetic construct, optionally contained within a suitable carrier, such as a cell, virus particle or liposome, amongst others.
  • a suitable carrier such as a cell, virus particle or liposome, amongst others.
  • the synthetic gene of the invention is inserted into a suitable vector or episome molecule, such as a bacteriophage vector, viral vector or a plasmid, cosmid or artificial chromosome vector which is capable of being maintained and/or replicated and/or expressed in the host cell, tissue or organ into which it is subsequently introduced.
  • a further aspect of the invention provides a genetic construct which at least comprises the synthetic gene according to any one or more of the embodiments described herein and one or more origins of replication and/or selectable marker gene sequences.
  • Genetic constructs are particularly suitable for the transformation of a eukaryotic cell to introduce novel genetic traits thereto, in addition to the provision of resistance characteristics to viral pathogens. Such additional novel traits may be introduced in a separate genetic construct or, alternatively on the same genetic construct which comprises the synthetic genes described herein.
  • additional novel traits may be introduced in a separate genetic construct or, alternatively on the same genetic construct which comprises the synthetic genes described herein.
  • an origin of replication or a selectable marker gene suitable for use in bacteria is physically-separated from those genetic sequences contained in the genetic construct which are intended to be expressed or transferred to a eukaryotic cell, or integrated into the genome of a eukaryotic cell.
  • the origin of replication is functional in a bacterial cell and comprises the pUC or the ColEl origin or alternatively the origin of replication is operable in a eukaryotic cell, tissue and more preferably comprises the 2 micron (2 ⁇ m) origin of replication or the SV40 origin of replication.
  • selectable marker gene includes any gene which confers a phenotype on a cell in which it is expressed to facilitate the identification and/or selection of cells which are transfected or transformed with a genetic construct of the invention or a derivative thereof.
  • Suitable selectable marker genes contemplated herein include the ampicillin-resistance gene (Amp r ), tetracycline-resistance gene (Tc r ), bacterial kanamycin-resistance gene (Kan r ), the zeocin resistance gene (Zeocin is a drug of bleomycin family which is trademark of InVitrogen Corporation), the AURI-C gene which confers resistance to the antibiotic aureobasidin A, phosphinothricin-resistance gene, neomycin phosphotransferase gene (nptil), hygromycin-resistance gene, ⁇ glucuronidase (GUS) gene, chloramphenicol acetyltransferase (CAT) gene, green fluorescent protein-encoding gene or the luciferase gene, amongst others.
  • Amicillin-resistance gene Amicillin-resistance gene
  • Tc r tetracycline-
  • the selectable marker gene is the nptil gene or Kan r gene or green fluorescent protein (GFP)-encoding gene.
  • selectable marker genes useful in the performance of the present invention and the subject invention is not limited by the nature of the selectable marker gene.
  • the present invention extends to all genetic constructs essentially as described herein, which include further genetic sequences intended for the maintenance and/or replication of said genetic construct in prokaryotes or eukaryotes and/or the integration of said genetic construct or a part thereof into the genome of a eukaryotic cell or organism.
  • Additional means for introducing recombinant DNA into plant tissue or cells include, but are not limited to, transformation using CaCI 2 and variations thereof, in particular the method described by Hanahan (1983), direct DNA uptake into protoplasts (Krens et al, 1982; Paszkowski et al, 1984), PEG-mediated uptake to protoplasts (Armstrong et al, 1990) microparticle bombardment, electroporation (Fromm et al., 1985), microinjection of DNA (Crossway et a., 1986), microparticle bombardment of tissue explants or cells (Christou et at, 1988; Sanford, 1988), vacuum-infiltration of tissue with nucleic acid, or in the case of plants, T-DNA-mediated transfer from Agrobacterium to the plant tissue as described essentially by An et al. (1985), Herrera-Estrella et a. (1983a, 1983b, 1985).
  • a microparticle is propelled into a cell to produce a transformed cell.
  • Any suitable ballistic cell transformation methodology and apparatus can be used in performing the present invention. Exemplary apparatus and procedures are disclosed by Stomp et al. (U.S. Pat. No. 5,122,466) and Sanford and Wolf (U.S. Pat. No. 4,945,050).
  • the genetic construct may incorporate a plasmid capable of replicating in the cell to be transformed.
  • microparticles suitable for use in such systems include 1 to 5 ⁇ m gold spheres.
  • the DNA construct may be deposited on the microparticle by any suitable technique, such as by precipitation.
  • the synthetic genes and genetic constructs described herein are adapted for integration into the genome of a cell in which it is expressed.
  • Those skilled in the art will be aware that, in order to achieve integration of a genetic sequence or genetic construct into the genome of a host cell, certain additional genetic sequences may be required. In the case of plants, left and right border sequences from the T-DNA of the Agrobacterium tumefaciens Ti plasmid will generally be required.
  • the present invention further extends to an isolated cell, tissue or organ comprising the synthetic gene described herein or a genetic construct comprising same.
  • the present invention extends further to regenerated tissues, organs and whole organisms derived from said cells, tissues and organs and to propagules and progeny thereof.
  • plants may be regenerated from transformed plant cells or tissues or organs on hormone-containing media and the regenerated plants may take a variety of forms, such as chimeras of transformed cells and non-transformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette), grafts of transformed and untransformed tissues (e.g., a transformed root stock grafted to an untransformed scion in citrus species).
  • Transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, a first generation (or T1) transformed plants may be selfed to give homozygous second generation (or T2) transformed plants, and the T2 plants further propagated through classical breeding techniques.
  • Adherent cell monolayers were grown in medium consisting of either DMEM (Life Technologies) supplemented with 10% v/v FBS (TRACE Biosciences or Life Technologies) or RPMI 1640 Medium (Life Technologies) supplemented with 10% v/v FBS. Cells were grown in incubators at 37° C. in an atmosphere containing 5% v/v CO 2 .
  • Tissue culture medium volumes were typically 192 ⁇ l/well for 96-well tissue culture plates, 360 ⁇ llwell for 48-well tissue culture plates, 3.8 ml/well for 6-well tissue culture plates, 9.6 ml for T25 and 39.2 ml for T75 tissue culture vessels.
  • Non-adherent cells were grown in growth medium similarly to adherent cell lines.
  • Tissue culture media volumes were typically 200 ⁇ l/well for 96-well tissue culture plates, 400 ⁇ l/well for 48-well tissue culture plates, 4 ml/well for 6-well tissue culture plates, 11 ml for T25 and 40 ml for T75 tissue culture vessels.
  • Adherent monolayers were washed twice with 1 x PBS and then treated with trypsin-EDTA for 5 min at 37° C.
  • Non-adherent cells were centrifuged for 5 min at 500 x g and 4° C.
  • the cells were suspended by trituration and transferred to storage medium consisting of DMEM or RPMI 1640 supplemented with 20% v/v FBS and 10% v/v dimethylsulfoxide (Sigma).
  • concentration of cells was determined by haemocytometer counting and diluted to 10 5 cells per ml. Aliquots were transferred to 1.5 ml cryotubes (Nunc) and the tubes were placed in a Cryo 1° C.
  • Freezing Container Naalgene containing propan-2-ol (BDH) and cooled slowly to ⁇ 70° C. The tubes of cells were then stored at ⁇ 70° C. Reanimation of frozen cells was achieved by warming the tubes to 0° C. on ice then transferring the cells to a T25 flask containing DMEM and 20% v/v FBS and incubating at 37° C. in an atmosphere of 5% v/v CO 2 .
  • Adherent and non-adherent mammalian cell lines were transfected with plasmid vectors containing expression constructs to target specific genes of interest. Stable, transformed colonies were selected over a period of 2-3 weeks using cell growth medium (either DMEM, 10% v/v FBS or RPMI 1640, 10% v/v FBS) supplemented with geneticin. Individual colonies were cloned to establish clonal lines of transfected cells.
  • cell growth medium either DMEM, 10% v/v FBS or RPMI 1640, 10% v/v FBS
  • Conditioned media were prepared by overlaying 20-30%-confluent monolayers of cells grown in a T75 vessel with 40 ml of DMEM, 10% v/v FBS. Vessels were incubated at 37° C. in 5% v/v CO 2 for 24 hr, after which the growth medium was transferred to a sterile 50 ml tube (Falcon) and centrifuged at 500 x g. The medium was passed through a 0.45 ⁇ m filter and decanted to a fresh sterile tube for use as conditioned medium.
  • Falcon sterile 50 ml tube
  • Non-adherent cells were cloned by dilution cloning.
  • Cell concentration was determined microscopically using a haemocytometer slide and cells were diluted to 10 cells per ml in conditioned medium.
  • Single wells of 96-well tissue culture vessels were seeded with 200 ⁇ l of the diluted cells and the plates were incubated at 37° C. in 5% v/v CO 2 for 48 hr.
  • Wells were inspected microscopically and those containing a single colony, arising from a single cell, were defined as clonal cell lines.
  • the medium was removed and replaced with 200 ⁇ l of fresh conditioned medium and cells incubated at 37° C. in 5% v/v CO 2 for 48 hr.
  • conditioned medium was replaced with 200 ⁇ l of DMEM, 10% v/v FBS and 1.5 mg/l genetecin and cells again incubated at 37° C. in 5% v/v CO 2. Colonies were allowed to expand in successive steps, with medium changes every 48 hr, until the stable, transformed lines were housed in T25 tissue culture vessels. At this point, aliquots of each stable cell line were frozen for long term maintenance
  • the tube was inverted several times to mix and the cells were collected by centrifugation at 500 x g for 10 min at 4° C. The supernatant was decanted and the pellet suspended in 5 ml of ice-cold 1 x PBS by gentle vortexing and a sample was counted ( ⁇ 2 ⁇ 10 8 ). The cells were collected by centrifugation at 500 x g for 10 min at 4° C. and the supernatant decanted.
  • the cell suspension is transferred into a 50 ml Falcon tube, centrifuged at 500 x g for 10 min at 4° C. and the supernatant decanted.
  • the pellet was suspended in 5 ml ice-cold 1 x PBS by gentle vortexing and the cells collected by centrifugation at 500 x g for 10 min at 4° C.
  • the supernatant was decanted and the pellet was resuspended in 5 ml of ice-cold 1 x PBS by gentle vortexing and a sample counted ( ⁇ 2 ⁇ 10 8 ).
  • the cells were collected by centrifugation at 500 x g for 10 min at 4° C. and the supernatant decanted.
  • Genomic DNA for both adherent and non-adherent cell lines, was extracted using the Qiagen Genomic DNA extraction kit (Cat No. 10243) according to the supplier's instructions. The concentration of genomic DNA was determined from absorbance at 260 nm using a Beckman model DU64 photospectrometer.
  • Genomic DNA (10 ⁇ g) was digested with appropriate restriction endonucleases and buffer in a volume of 200 ⁇ l at 37° C. for approximately 16 hr. Following digestion, 20 ⁇ l of 3M sodium acetate, pH 5.2, and 500 ⁇ l of absolute ethanol were added to the digest and the solution mixed by vortexing and chilled at ⁇ 20° C. for 2 hr. DNA was recovered by centrifugation at 10,000 x g for 30 min at 4° C. The supernatant was removed and the DNA pellet rinsed with 500 ⁇ l of 70% v/v ethanol, the pellet air-dried and the DNA dissolved in 20 ⁇ l of water.
  • the gel was soaked in 1.5 M NaCI, 0.5 M NaOH then in 1.5 M NaCI, 0.5 M Tris-HOCI, pH 7.0.
  • the DNA fragments were then capillary blotted to Hybond NX (Amersham) membrane and fixed by UV cross-linking (Bio Rad GS Gene Linker).
  • Hybond membrane was rinsed in sterile water and stained in 0.4% v/v methylene blue in 300mM sodium acetate, pH 5.2, for 5 min to visualize the transferred genomic DNA. The membrane was then rinsed twice in sterile water, destained in 40% v/v ethanol then rinsed in sterile water.
  • the membrane was placed in a Hybaid bottle with 5 ml of pre-hybridization solution (6 x SSPE, 5 x Denhardt's reagent, 0.5% w/v SDS, 100 ⁇ g/ml denatured, fragmented herring sperm DNA) and pre-hybridized at 60° C. for approximately 14 hr in a hybridization oven with constant rotation (6 rpm).
  • pre-hybridization solution 6 x SSPE, 5 x Denhardt's reagent, 0.5% w/v SDS, 100 ⁇ g/ml denatured, fragmented herring sperm DNA
  • Probe 25 ng was labelled with [ ⁇ 32 P]-dCTP (specific activity 3000 Ci/mmol) using the Megaprime DNA labelling system as per the supplier's instructions (Amersham Cat. No. RPN1606). Labelled probe was passed through a G50 Sephadex Quick Spin (trademark) column (Roche, Cat. No. 1273973) to remove unincorporated nucleotides as per the supplier's instructions.
  • the heat-denatured labelled probe was added to 2 ml of hybridization buffer (6 x SSPE, 0.5% w/v SDS, 100 ⁇ g/ml denatured, fragmented herring sperm DNA) pre-warmed to 60° C.
  • the pre-hybridization buffer was decanted and replaced with 2 ml of pre-warmed hybridization buffer containing the labelled probe.
  • the membrane was hybridized at 60° C. for approximately 16 hr in a hybridization oven with constant rotation (6 rpm).
  • Washing duration at 68° C. varied based on the amount of radioactivity detected with a hand-held Geiger counter.
  • the damp membrane was wrapped in plastic wrap and exposed to X-ray film (Curix Blue HC-S Plus, AGFA) for 24-48 hr and the film developed to visualize bands of probe hybridized to genomic DNA.
  • X-ray film Curix Blue HC-S Plus, AGFA
  • Cover slips were mounted on glass microscope slides, three to a slide, in glycerol/DABCO (25 mg/ml DABCO (1,4-diazabicyclo (2.2.2)octane (Sigma D 2522)) in 80% v/v glycerol in PBS) and examined with a 100x oil immersion objective under UV illumination at 500-550 nm.
  • glycerol/DABCO 25 mg/ml DABCO (1,4-diazabicyclo (2.2.2)octane (Sigma D 2522)
  • Plasmid pBluescriptII (SK+) is commercially available from Stratagene and comprises the LacZ promoter sequence and lacZ-alpha transcription terminator, with a multiple cloning site for the insertion of structural gene sequences therein.
  • the plasmid further comprises the ColE1 and f1 origins of replication and ampicillin-resistance gene.
  • Plasmid pSVL is commercially-obtainable from Pharmacia and serves as a source of the SV40 late promoter sequence.
  • the nucleotide sequence of pSVL is also publicly available as GenBank Accession Number U13868.
  • Plasmid pCR2.1 is commercially available from Invitrogen and comprises the LacZ promoter sequence and lacZ- ⁇ transcription terminator, with a cloning site for the insertion of structural gene sequences there between. Plasmid pCR2.1 is designed to clone nucleic acid. fragments by virtue of the A-overhang frequently synthesized by Taq polymerase during the polymerase chain reaction. PCR fragments cloned in this fashion are flanked by two EcoRI sites. The plasmid further comprises the ColE1 and f1 origins of replication and kanamycin-resistance and ampicillin-resistance genes.
  • Plasmid pEGFP-N1 MCS (FIG. 1; Olontech) contains the CMV IE promoter operably connected to an open reading frame encoding a red-shifted variant of wild-type green fluorescent protein (GFP; Prasher et al, 1992; Chalfie et aL, 1994; Inouye and Tsuji, 1994), which has been optimised for brighter fluorescence.
  • GFP wild-type green fluorescent protein
  • the specific GFP variant encoded by pEGFP-N1 MCS has been disclosed by Cormack et al. (1996).
  • Plasmid pEGFP-N1 MCS contains a multiple cloning site comprising Bg/ll and BamHl sites and many other restriction endonuclease cleavage sites, located between the CMV IE promoter and the GFP open reading frame.
  • Structural genes cloned into the multiple cloning site will be expressed at the transcriptional level if they lack a functional translation start site, however such structural gene sequences will not be expressed at the protein level (i.e. translated).
  • Structural gene sequences inserted into the multiple cloning site which comprise a functional translation start site will be expressed as GFP fusion polypeptides if they are cloned in-frame with the GFP-encoding sequence.
  • the plasmid further comprises an SV40 polyadenylation signal downstream of the GFP open reading frame to direct proper processing of the 3′-end of mRNA transcribed from the CMV-IE promoter sequence.
  • the plasmid further comprises the SV40origin of replication functional in animal cells; the neomycin-resistance gene comprising SV40 early promoter (SV40 EP in FIG. 1) operably connected to the neomycin/kanamycin-resistance gene derived from Tn5 (Kan/neo in FIG. 1) and the HSV thymidine kinase polyadenylation signal (HSV TK poly (A) in FIG.
  • Plasmid pCMV.cass (FIG. 2) is an expression cassette for driving expression of a structural gene sequence under control of the CMV-IE promoter sequence. Plasmid pCMV.cass was derived from pEGFP-N1 MCS by deletion of the GFP open reading frame as follows: Plasmid pEGFP-N1 MCS was digested with PinAl and Notl blunt-ended using Pful polymerase and then re-ligated. Structural gene sequences are cloned into pCMV.cass using the multiple cloning site, which is identical to the multiple cloning site of pEGFP-N1 MCS, except it lacks the PinAl site.
  • Plasmid pCMV.SV40L.cass (FIG. 3) comprises the synthetic poly A site and the SV40 late promoter sequence from plasmid pCR.SV40L (FIG. 4), sub-cloned as a Sal I fragment, into the Sal I site of plasmid pCMV.cass (FIG. 2), such that the CMV-IE promoter and SV40 late promoter sequences are capable of directing transcription in the same direction.
  • the synthetic poly(A) site at the 5′end of the SV40 promoter sequence is used as a transcription terminator for structural genes expressed from the CMV IE promoter in this plasmid, which also provides for the insertion of said structural gene via the multiple cloning site present between the SV40 late promoter and the synthetic poly(A) site (FIG. 5).
  • the multiple cloning sites are located behind the CMV-IE and SV40 late promoters, including BamHl and BgAl sites.
  • Plasmid pCMV.SV40LR.cass (FIG. 4) comprises the SV40 late promoter sequence derived from plasmid pCR.SV40L, sub-cloned as a SaAl fragment into the SaA site of the plasmid pCMV.cass (FIG. 2), such that the CMV-IE or the SV40 late promoter may drive transcription of a structural gene or a multiple structural gene unit, in the sense or antisense orientation, as desired.
  • a multiple cloning site is positioned between the opposing CMV-IE and SV40 late promoter sequences in this plasmid to facilitate the introduction of a structural gene sequence.
  • the structural gene sequence or multiple structural gene unit which is to be introduced into pCMV,SV40LR.cass will comprise both a 5′and a 3′polyadenylation signal as follows:
  • suitably-oriented terminator sequences may be placed at the 5′-end of the CMV and SV40L promoters, as shown in FIG. 4.
  • plasmid pCMV.SV40LR.cass is further modified to produce a derivative plasmid which comprises two polyadenyfation signals located between the CMV IE and SV40 late promoter sequences, in approriate orientations to facilitate expression of any structural gene located therebetween in the sense or antisense orientation from either the CMV IE promoter or the SV40 promoter sequence.
  • the present invention clearly encompasses such derivatives.
  • Plasmid pCR.Bgt-GFP-Bam (FIG. 5) comprises an internal region of the GFP open reading frame derived from plasmid pEGFP-N1 MCS (FIG. 1) placed operably under the control of the lacZ promoter.
  • a region of the GFP open reading frame was amplified from pEGFP-N1 MCS using the amplification primers Bgl-GFP (SEQ ID NO:1) and GFP-Bam (SEQ ID NO:2) and cloned into plasmid pCR2.1.
  • the internal GFP-encoding region in plasmid pCR.Bgl-GFP-Bam lacks functional translational start and stop codons.
  • EGFP (FIG. 6) comprises the EGFP open reading frame derived from plasmid pEGFP-N1 MCS (FIG. 1) placed operably under the control of the lacZ promoter.
  • the EGFP encoding region of pEGFP-N1 MCS was excised as a NotllXhol fragment and cloned into the Notl/Xhol cloning sites of plasmid pBluescript 11 (SK+).
  • Plasmid pCMV.EGFP (FIG. 7) is capable of expressing the EGFP structural gene under the control of the CMV-IE promoter sequence.
  • the EGFP sequence from plasmid pBSll(SK+).EGFP was excised as BamHI/Sacl fragment and cloned into the Bgil/Sacl sites of plasmid pCMV.cass (FIG. 2).
  • Plasmid pCR.SV40L (FIG. 8) comprises the SV40 late promoter derived from plasmid pSVL (GenBank Accession No. U13868; Pharmacia), cloned into pCR2.1 (Stratagene).
  • the SV40 late promoter was amplified using the primers SV40-1 (SEQ ID NO:3) and SV40-2 (SEQ ID NO:4) which comprise Sal I cloning sites to facilitate sub-cloning of the amplified DNA fragment into pCMV.cass.
  • the primer also contains a synthetic poly (A) site at the 5′end, such that the amplicification product comprises the synthetic poly(A) site at the 5′end of the SV40 promoter sequence.
  • the BEV RNA-dependent RNA polymerase coding region was amplified as a 1,385 bp DNA fragment from a full-length cDNA clone encoding same, using primers designated BEV-1 (SEQ ID NO:5) and BEV-2 (SEQ ID NO:6), under standard amplification conditions.
  • the amplified DNA contained a 5′-Bgl ll restriction enzyme site, derived from the BEV-1 primer sequence and a 3′BamHl restriction enzyme site, derived from the BEV-2 primer sequence.
  • the amplified BEV polymerase structural gene comprises the start site in-frame with BEV polymerase-encoding nucleotide sequences,
  • the amplified BEV polymerase structural gene comprises the ATG start codon immediately upstream (ie. juxtaposed) to the BEV polymerase-encoding sequence.
  • the complete BEV polymerase coding region was amplified from a full-length CDNA clone encoding same, using primers BEV-1 and BEV-3.
  • Primer BEV-3 comprises a BamHI restriction enzyme site at positions 5 to 10 inclusive and the complement of a translation stop signal at positions 11 to 13.
  • an open reading frame comprising a translation start signal and translation stop signal, contained between the Bgl II and BamHl restriction sites.
  • the amplified fragment was cloned into pCR2.1 (Stratagene) to produce plasmid pCR2.BEV.2 (FIG. 10).
  • a non-translatable BEV polymerase structural gene was amplified from a full-length BEV polymerase CDNA clone using the amplification primers BEV-3 (SEQ ID NO:7) and BEV-4 (SEQ ID NO:8).
  • Primer BEV-4 comprises a BglI cloning site at positions 5-10 and sequences downstream of this Bg/II site are homologous to nucleotide sequences of the BEV polymerase gene.
  • the BEV polymerase is expressed as part of a polyprotein and, as a consequence, there is no ATG translation start site in this gene.
  • the amplified DNA was cloned into plasmid pCR2.1 (Stratagene) to yield plasmid pCR.BEV.3 (FIG. 11).
  • Plasmid pCMV.EGFP.BEV2 (FIG. 12) was produced by cloning the BEV polymerase sequence from pCR.BEV.2 as a Bglll/BamHl fragment into the BamHl site of pCMV.EGFP.
  • Plasmid pCMV.BEV.2 (FIG. 13) is capable of expressing the entire BEV polymerase open reading frame under the control of CMV-IE promoter sequence.
  • the BEV polymerase sequence from pCR.BEV.2 was sub-cloned in the sense orientation as a Bg/ll-to-BamHl fragment into Bg/ll/BamHl-digested pCMV.cass (FIG. 2).
  • Plasmid pCMV.BEV.3 (FIG. 14) expresses a non-translatable BEV polymerase structural gene in the sense orientation under the control of the CMV-IE promoter sequence.
  • the BEV polymerase sequence from pCR.BEV.3 was sub-cloned in the sense orientation as a Bg/ll-to-BamHl fragment into Bg/ll/BamHl-digested pCMV.cass (FIG. 2).
  • Plasmid pCMV.VEB (FIG. 15) expresses an antisense BEV polymerase mRNA under the control of the CMV-lE promoter sequence.
  • the BEV polymerase sequence from pCR.BEV.2 was sub-cloned in the antisense orientation as a Bg/ll-to-BamrHI fragment into Bg/ll-BamHl-digested 'pCMV.cass (FIG. 2).
  • Plasmid pCMV.BEV.GFP (FIG. 16) was constructed by cloning the GFP fragment from pCR.Bgl-GFP-Bam as a Bg/ll/BamHl fragment into BamHl-digested pCMV.BEV.2. This plasmid serves as a control in some experiments and also as an intermediate construct.
  • Plasmid pCMV.BEV.SV40L.0 (FIG. 17) comprises a translatable BEV polymerase structural gene derived from plasmid pCR.BEV.2 inserted in the sense orientation between the CMV-lE promoter and the SV40 late promoter sequences of plasmid pCMV.SV40L.cass.
  • the BEV polymerase structural gene was sub-cloned as a BgAl-to-BamHl fragment into Bg/ll-digested pCMV.SV40L.cass DNA.
  • Plasmid pCMV.O.SV40L.BEV (FIG. 18) comprises a translatable BEV polymerase structural gene derived from plasmid pCR.BEV.2 cloned downstream of tandem CMV-lE promoter and SV40 late promoter sequences present in plasmid pCMV.SV40L.cass.
  • the BEV polymerase structural gene was sub-cloned in the sense orientation as a Bg/ll-to-BamHI fragment into BamHl-digested pCMV.SV40L.cass DNA.
  • Plasmid pCMV.O.SV40L.VEB (FIG. 19) comprises an antisense BEV polymerase structural gene derived from plasmid pCR.BEV.2 cloned downstream of tandem CMV-lE promoter and SV40 late promoter sequences present in plasmid pCMV.SV40L.cass.
  • the BEV polymerase structural gene was sub-cloned in the antisense orientation as a Bg/ll-to-BamHI fragment into BamHl-digested pCMV.SV40L.cass DNA.
  • Plasmid pCMV.BEVx2 (FIG. 20) comprises a direct repeat of a complete BEV polymerase open reading frame under the control of the CMV-lE promoter sequence. In eukaryotic cells at least, the open reading frame located nearer the CMV-lE promoter is translatable.
  • the BEV polymerase structural gene from plasmid pCR.BEV.2 was sub-cloned in the sense orientation as a BgAl-to-BamHl fragment into BamHi-digested pCMV.BEV.2, immediately downstream of the BEV polymerase structural gene already present therein.
  • Plasmid pCMV.BEVx3 (FIG. 21) comprises a direct repeat of three complete BEV polymerase open reading frames under the control of the CMV-1 E promoter.
  • the BEV polymerase fragment from pCR.BEV.2 was cloned in the sense orientation as a BgAilBamHl fragment into the BamHl site of pCMV.BEVx2, immediately downstream of the BEV polymerase sequences already present therein.
  • Plasmid pCMV.BEVx4 (FIG. 22) comprises a direct repeat of four complete BEV polymerase open reading frames under the control of the CMV-1 E promoter.
  • the BEV polymerase fragment from pCR.BEV.2 was cloned in the sense orientation as a BgIll/BamHf fragment into the BamHI site of pCMV.BEVx3, immediately downstream of the BEV polymerase sequences already present therein.
  • Plasmid pCMV.BEV.SV40L.BEV(FIG. 23) comprises a multiple structural gene unit comprising two BEV polymerase structural genes placed operably and separately under control of the CMV-lE promoter and SV40 late promoter sequences.
  • the translatable BEV polymerase structural gene present in pCR.BEV.2 was sub-cloned in the sense orientation as a BgIll-to-BamHI fragment behind the SV40 late promoter sequence present in BamHi-digested pCMV.BEV.SV40L-O.
  • Plasmid pCMV.BEV.SV40L.VEB (FIG. 24) comprises a multiple structural gene unit comprising two BEV polymerase structural genes placed operably and separately under control of the CMV-lE promoter and SV40 late promoter sequences.
  • the translatable BEV polymerase structural gene present in pCR.BEV.2 was sub-cloned in the antisense orientation as a Bg/ll-to-BamHI fragment behind the SV40 late promoter sequence present in BamHl-digested pCMV.BEV.SV4OL-O.
  • the BEV polymerase structural gene is expressed in the sense orientation under control of the CMV-lE promoter to produce a translatable mRNA, whilst the BEV polymerase structural gene is also expressed under control of the SV40 promoter to produce an antisense mRNA species.
  • Plasmid pCMV.BEV.GFP.VEB (FIG. 25) comprises a BEV structural gene inverted repeat or palindrome, interrupted by the insertion of a GFP open reading frame (stuffer fragment) between each BEV structural gene sequence in the inverted repeat.
  • the GFP stuffer fragment from pCR.BgI-GFP-Bam was first sub-cloned in the sense orientation as a BgAl-to-BamHl fragment into BamHi-digested pCMV.BEV.2 to produce an intermediate plasmid pCMV.BEV.GFP wherein the BEV polymerase-encoding and GFP-encoding sequences are contained within the same 5′-Bg/ll-to-BamHI-3′fragment.
  • the BEV polymerase structural gene from pCMV.BEV.2 was then cloned in the antisense orientation as a Bg/ll-to-BamHI fragment into BamHl-digested pCMV.BEV.GFP.
  • the BEV polymerase structural gene nearer the CMV-lE promoter sequence in plasmid pCMV.BEV.GFP.VEB is capable of being translated, at least in eukaryotic cells.
  • Plasmid pCMV.EGFP.BEV2.PFG (FIG. 26) comprise a GFP palindrome, interrupted by the insertion of a BEV polymerase sequence between each GFP structural gene in the inverted repeat.
  • the GFP fragment from pCR.BgI-GFP-Bam was cioned as a Bg/ll/BamHI fragment into the BamHI site of pCMV.EGFP.BEV2 in the antisense orientation relative to the CMV promoter.
  • Plasmid pCMV.BEV.SV40LR (FIG. 27) comprises a structural gene comprising the entire BEV polymerase open reading frame placed operably and separately under control of opposing CMV-lE promoter and SV40 late promoter sequences, thereby potentially producing BEV polymerase transcripts at least from both strands of the fun-length BEV polymerase structural gene.
  • the translatable BEV polymerase structural gene present in pCR.BEV.2 was sub-cloned, as a BgAl-to-BamHl fragment, into the unique Bgni site of plasmid pCMV.SV40LR.cass, such that the BEV open reading frame is present in the sense orientation relative to the CMV-lE promoter sequence.
  • Plasmid pcDNA3 is commercially available from Invitrogen and comprises the CMV-lE promoter and BGHpA transcription terminator, with multiple cloning sites for the insertion of structural gene sequences there between.
  • the plasmid further comprises the ColE1 and fl origins of replication and neomycin-resistance and ampicillin-resistance genes.
  • Plasmid pcDNA3.Galt (BresaGen Limited, South Australia, Australia; FIG. 28) is plasmid pcDNA3 (Invitrogen) and comprises the cDNA sequence encoding porcine gene alpha-1,3-galactosyltransferase (Galt) operably under the control of the CMV-lE promoter sequence such that it is capable of being expressed therefrom.
  • porcine gene alpha-1,3-galactosyltransferase cDNA was cloned as an EcoRl fragment into the EcoRl cloning site of pcDNA3.
  • the plasmid further comprises the ColE1 and fl origins of replication and the neomycin and ampicillin-resistance genes.
  • Plasmid pCMV.Galt (FIG. 29) is capable of expressing the Galt structural gene under the control of the CMV-lE promoter sequence.
  • the Galt sequence from plasmid pcDNA3.Galt was excised as an EcoRl fragment and cloned in the sense orientation into the EcoRi site of plasmid pCMV.cass (FIG. 2).
  • Plasmid pCMV.EGFP.Galt (FIG. 30) is capable of expressing the Galt structural gene as a Galt fusion polypeptide under the control of the CMV-lE promoter sequence.
  • the Galt sequence from pCMV.GaIt (FIG. 29) was excised as a Bg/lllBamHl fragment and cloned into the BamHl site of pCMV.EGFP.
  • Plasmid pCMV.Galt.GFP (FIG. 31) was produced by cloning the Galt cDNA as an EcORI fragment from pCDNA3 into EcoRi-digested pCMV.EGFP in the sense orientation. This plasmid serves as both a control and construct intermediate. Plasmid pCMV.Galt-SV40L.0
  • the plasmid pCMV.Galt.SV40L0 (FIG. 32) comprises a Galt structural gene cloned downstream of the CMV promoter present in pCMV.SV40L.cass.
  • the Galt cDNA fragment from pCMV.GaIt was cloned as a 'Bg/ll/BamHl into Bg/ll-digested pCMV.SV40L.cass in the sense orientation.
  • the plasmid pCMV.O.SV40L.tlaG (FIG. 33) comprises a Galt structural gene clones in an antisense orientation downstream of the SV40L promoter present in pCMV.SV40L.cass.
  • the Galt CDNA fragment from pCMV.Galt was cloned as a Bg/ll/BamHl into BamHl-digested pCMV.SV40L.cass in the antisense orientation.
  • the plasmid pCMV.O.SV40L.Galt (FIG. 34) comprises a Galt structural gene cloned downstream of the SV40L promoter present in pCMV.SV40L.cass.
  • the Galt cDNA fragment from pCMV.Galt was cloned as a Bg/ll/BamHl into BamHi-digested pCMV.SV40L.cass in the sense orientation.
  • Plasmid pCMV.Galtx2 (FIG. 35) comprises a direct repeat of a Galt open reading frame under the control of the CMV-lE promoter sequence. In eukaryotes cells at least, the open reading frame located nearer the CMV-lE promoter is translatable.
  • the Galt structural gene from pCMV.Galt was excised as a Bg/ll/BamHl fragment and cloned in the sense orientation into the BamHl cloning site of pCMV.Galt.
  • Plasmid pCMV.Galtx4 (FIG. 36) comprises a quadruple direct repeat of a Galt open reading frame under the control of the CMV-lE promoter sequence. In eukaryotes cells at least, the open reading frame located nearer the CMV-lE promoter is translatable.
  • the Galtx2 sequence from pCMV.Galtx2 was excised as a Bg/ll/BamHl fragment and cloned in the sense orientation into the BamHI cloning site of pCMV.Galtx2.
  • the plasmid pCMV.Galt.SV40L.Galt (FIG. 37) is designed to express two sense transcripts of Galt, one driven by the CMV promoter, the other by the SV40L promoter.
  • a Galt cDNA fragment from pCMV.Galt was cloned as a Bg/ll/BamHl fragment into Bg/ll-digested pCMV.O.SV40.Galt in the sense orientation.
  • the plasmid pCMV.Galt.SV40.tlaG (FIG. 38) is designed to express a sense transcript of Galt driven by the CMV promoter and an antisense transcript driven by the SV40L promoter.
  • a Galt cDNA fragment from pCMV.Galt was cloned as a Bglll/BamHl fragment into Bglll-digested pCMV.O.SV40.taIG in the sense orientation.
  • Plasmid pCMV.Galt.GFP.tiaG (FIG. 39) comprise a Galt palindrome, interrupted by the insertion of a GFP sequence between each Galt structural gene in the inverted repeat.
  • the Bg/ll/BarnHl Galt cDNA fragment from pCMV.Galt was cloned into the BamHl site of pCMV.GaIt.GFP in the antisense relative to the CMV promoter.
  • the plasmid pCMV.EGFP.Galt.PFG (FIG. 40) comprises a GFP palindrome, interrupted by the insertion of a Galt sequence between each GFP structural gene of the inverted repeat, expression of which is driven by the CMV promoter.
  • the Galt sequences from pCMV.Galt were cloned as a Bglll/BamHl fragment into BamHl-digested pCMV.EGFP in the sense orientation to produce the intermediate pCMV.EGFP.Galt (not shown); following this further GFP sequences from pCR.Bgl-pCMV.EGFP.Galt in the antisense orientation.
  • the plasmid pCMV.Galt.SV4GLR (FIG. 41) is designed to express Galt cDNA sequences cloned between the opposing CMV and SV40L promoters in the expression cassette pCMV.SV40LR.cass.
  • Galt sequences from pCMV.Galt were cloned as a Bgll/BamHl fragment in Bglll-digested pCMV.SV40LR.cass in the sense orientation relative to the 35S promoter.
  • Plasmid pART27 is a binary vector, specifically designed to be compatible with the pART7 expression cassette. It contains bacterial origins of replication for both E. coil and Agrobacterium tumefaciens , a spectinomycin resistance gene for bacterial selection, left and right T-DNA borders for transfer of DNA from Agrobacterium to plant cells and a kanamycin resistance cassette to permit selection of transformed plant cells.
  • the kanamycin resistance cassette is located between the T-DNA borders
  • pART27 also contains a unique Notl restriction site which permits cloning of constructs prepared in vectors such as pART7 to be cloned between the T-DNA borders. Construction of pART27 is described in Gleave, AP (1992).
  • Plasmid pBC (KS-) is commercially available from Stratagene and comprises the
  • LacZ promoter sequence and lacZ-alpha transcription terminator with a multiple cloning site for the insertion of structural gene sequences therein.
  • the plasmid further comprises the ColEl arid fl origins of replication and a chloroamphenicol-resistance gene.
  • Plasmid pSP72 is commercially available from Promega and contains a multiple cloning site for the insertion of structural gene sequences therein.
  • the plasmid further comprises the ColE1 origin of replication and an ampicillin-resistance gene.
  • Plasmid pART7 is an expression cassette designed to drive expression of sequences cloned behind the 35S promoter. It contains a polylinker to assist cloning and a region of the octipine synthase terminator.
  • the 35S expression cassette is flanked by two Not I restriction sites which permits cloning into binary expression vectors, such as pART27 which contains a unique NotI site. Its construction as described in Gleave, AP (1992), a map is shown in FIG. 42,
  • Plasmid p35S.CMV.cass was designed to express two separate gene sequences cloned into a single plasmid. To create this plasmid, sequences corresponding to the nos terminator and the SCBV promoter were amplified by PCR then cloned in the polylinker of pART7 between the 35S promoter and OCS.
  • the resulting plasmid has the following arrangement of elements:
  • 35S promoter polylinker 1 — NOS terminator — SCBV promoter - polylinker 2-OCS terminator.
  • NOS terminator sequences were amplified from the plasmid pAHC27 (Christensen and Quail, 1996) using the two oligonucleotides;
  • Nucleotide residues 1 to 17 for NOS 5′and 1 to 15 for NOS 3′ represent additional nucleotides designed to assist in construct preparation by adding additional restriction sites.
  • NOS 5′these are BamHl, Smal, Aatll and the first 4 bases of an Nrul site
  • NOS 3′these are Ncol and Sfil sites.
  • the remaining sequences for each oligonucleotide are homologous to the 5′and 3′ends respectively of NOS sequences in pAHC 27.
  • SCBV promoter sequences were amplified from the plasmid pScBV-20 (Tzafir et at, 1998) using the two oligonucleotides:
  • SCBV 5′ 5′-CCATGGCCTATATGGCCATTCCCCACATTCAAG-3′(SEQ ID NO:1 1);
  • SCBV 3′ 5′-AACGTTAACTTCTACCCAGTTCCAGAG-3′(SEQ ID NQ:12)
  • a region of the PVY genome was amplified by PCR using reverse-transcribed RNA isolated from PVY-infected tobacco as a template using standard protocols and cloned into a plasmid pGEM 3 (Stratagene), to create pGEM.PVY.
  • pGEM 3 (Stratagene)
  • a Sall/Hindlll fragment from pGEM.PVY, corresponding to a Sall/Hindlll fragment positions 1536-2270 of the PVY strain O sequence was then subcloned into the plasmid pBC (Stratagene Inc.) to create pBC.PVY (FIG. 44).
  • Plasmid pSP72.PVY was prepared by inserting an EcoRl/Sall fragment from pBC.PVY into EcoRi/Sall cut pSP72 (Promega). This construct contains additional restriction sites flanking the PVY insert which were used to assist subsequent manipulations. A map of this construct is shown in FIG. 45.
  • Plasmid ClapBC.PVY was prepared by inserting a Clal/Sall fragment from pSP72.PVY into Clal/Sal I cutpBC (Stratagene). This construct contains additional restriction sites flanking the PVY insert which were used to assist subsequent manipulations. A map of this construct is shown in FIG. 46.
  • Plasmid pBC.PVYx2 contains two direct head-to-tail repeats of the PVY sequences derived from pBC.PVY.
  • the plasmid was generated by cloning an Accf/Clal PVY fragment from pSP72.PVY into Acci cut pBC.PVY and is shown in FIG. 47.
  • Plasmid pSP72.PVYx2 contains two direct head-to-tail repeats of the PVY sequences derived from pBC.PVY.
  • the plasmid was generated by cloning an Accl/Clal PVY fragment from pBc.PVY into Accl cut pSP72.PVY and is shown in FIG. 48.
  • Plasmid pBC.PVYx3 contains three direct head-to-tail repeats of the PVY sequences derived from pBC.PVY.
  • the plasmid was prepared by cloning an Accl/Clal PVY fragment from pSP72.PVY into Accl cut pBC.PVYx2 and is shown in FIG. 49.
  • Plasmid pBC.PVYx4 contains four direct head-to-tail repeats of the PVY sequences derived from pBC.PVY.
  • the plasmid was prepared by cloning the direct repeat of PVY sequences from pSP72.PVYx2 as an Accl/Clal fragment into Acci cut pBC.PVYx2 and is shown in FIG. 50.
  • the first 9 nucleotide of these primers encode a BamHl site, the remaining nucleotides are homologous to sequences of the LNYV 4b gene.
  • the fragment was cloned into the EcoRl site of pCR2.1 (Stratagene). This EcoRl fragment was cloned into the EcoRl site of Cia pBC.PVY to create the intermediate plasmid pBC.PVY.LNYV which is shown in FIG. 51.
  • the plasmid pBC.PVY.LNYV.YVP contains an interrupted direct repeat of PVY sequences.
  • a Hpal/Hincil fragment from pSP72 was cloned into Smal-digested pBC.PVY.LNYV and a plasmid containing the sense orientation isolated, a map of this construct is shown in FIG. 52.
  • the plasmid pBV.PVY.LNYV.YVPL contains a partial interrupted palindrome of PVY sequences.
  • One arm of the palindrome contains all the PVY sequences from pBC.PVY, the other arm contains part of the sequences from PVY, corresponding to sequences between the EcoRV and Hincli sites of pSP72.PVY.
  • To create this plasmid an EcoRV/Hincli fragment from pSP72.PVY was cloned into Smal-digested pBC.PVY.LNYV and a plasmid containing the desired orientation isolated, a map of this construct is shown in FIG. 53.
  • the plasmid pBC.PVY.LNYV.YVP contains an interrupted palindrome of PVY sequences.
  • a Hpal/Hincil fragment from pSP72. was cloned into Sma-digested pBC.PVY.LNYV and a plasmid containing the antisense orientation isolated, a map of this construct is shown in FIG. 54.
  • Plasmid pART7.PVY (FIG. 55) was designed to express PVY sequences driven by the 35S promoter. This plasmid serves as a control construct in these experiments, against which all other constructs was compared. To generate this plasmid a Clal/Accl fragment from ClapBC.PVY was cloned into Clal-digested pART7 and a plasmid with expected to express a sense PVY sequence with respect to the PVY genome, was selected.
  • Plasmid pART7.35S.PVY.SCBV.O (FIG. 56) was designed to act as a control for co-expression of multiple constructs from a single plasmid in transgenic plants.
  • the 35S promoter was designed to express PVY sense sequences, whilst the SCBV promoter was empty.
  • the PVY fragment from Cla pBC.PVY was cloned as a Xhol/EcoRl fragment into Xhol/EcoRl-digested pART7.35S.SCBV.cass to create p35S.PVY.SCBV.O.
  • Plasmid pART27.35S.O.SCBV.PVY (FIG. 57) was designed to act as an additional control for co-expression of multiple constructs from a single plasmid in transgenic plants. No expressible sequences were cloned behind the 35S promoter, whilst the SCBV promoter drove expression of a PVY sense fragment. To generate this plasmid, the PVY fragment from Cla pBC.PVY was cloned as a CIal fragment into Clal-digested pART7.35S.SCBV.cass, a plasmid containing PVY sequences in a sense orientation was isolated and designated p35S.O.SCBV.PVY.
  • Plasmid pART7.35S.O.SCBV.YVP (FIG. 58) was designed to act as an additional control for co-expression of multiple constructs from a single plasmid in transgenic plants. No expressible sequences were cloned behind the 35S promoter, whilst the SCBV promoter drove expression of a PVY antisense fragment.
  • the PVY fragment from Cla pBC.PVY was cloned as a Clal fragment into Clai-digested p35S.SCBV.cass, a plasmid containing PCY sequences in an antisense orientation was isolated and designated p35S.O.SCBV.YVP.
  • Plasmid pART7.PVYx2 (FIG. 59) was designed to express a direct repeat of PVY sequences driven by the 35S promoter in transgenic plants. To generate this plasmid, direct repeats from pBC.PVYx2 were cloned as a Xhol/BamHl fragment into Xhol/BamHl cut pART7. Sequences consisting of the 35 S promoter, direct repeats of PVY and the OCS terminator were excised as a Notl fragment from pART7.PVYx2 and cloned into Notl-digested pART27, a plasmid with the desired insert orientation was selected and designated pART27.PVYx2.
  • Plasmid pART7.PVYx3 (FIG. 60) was designed to express a direct repeat of three PVY sequences driven by the 35S promoter in transgenic plants. To generate this plasmid, direct repeats from pBC.PVYx3 were cloned as a Xhol/BamHl fragment into XhoflBamHl cut pART7. Sequences consisting of the 35S promoter, direct repeats of PVY and OCS terminator were excised as a Notl fragment from pART.PVYx3 and cloned into Notl-digested pART27, a plasmid with the desired insert orientation was selected and designated pART27.PVYx3.
  • Plasmid pART7.PVYx4 (FIG. 61) was designed to express a direct repeat of four PVY sequences driven by the 355 promoter in transgenic plants. To generate this plasmid, direct repeats from pBC.PVYx4 were cloned as a Xhol/BamHl fragment into xhol/BamHi cut pART7. Sequences consisting of the 35S promoter, direct repeats of PVY and the OCS terminator were excised as a Notl fragment from pART7.PVYx3 and cloned into Notl-digested pART27, a plasmid with the desired insert orientation was selected and designated pART27.PVYx3.
  • Plasmid pART7.PVY.LNYV.PVY (FIG. 62) was designed to express the interrupted direct repeat of PVY sequences driven by the 35S promoter in transgenic plants. This construct was prepared by cloning the interrupted direct repeat of PVY from pBC.PVY.LNYV.PVY as a Xhol/Xbal fragment into pART7 digested with Xhol/Xbal.
  • Plasmid pART7.PVY.LNYV.YVPa (FIG. 63) was designed to express the partial interrupted palindrome of PVY sequences driven by the 35S promoter in transgenic plants. This construct was prepared by cloning the partial interrupted palindrome of PVY sequences from pBC.PVY.LNYV.YVP ⁇ as a Xhol/Xbal fragment into pART7 digested with Xhol/Xbal.
  • Plasmid pART7.PVY.LNYV.YVP (FIG. 64) was designed to express the interrupted palindrome of PVY sequences driven by the 35S promoter in transgenic plants. This construct was prepared by cloning the interrupted palindrome of PVY sequences from pBC.PVY.LNYV.YVP ⁇ as a Xhol/Xbal fragment into pART7 digested with Xho)lIXbal.
  • Plasmid pART7.35S.PVY.SCBV.YVP (FIG. 65) was designed to co-express sense and antisense constructs in transgenic plants. To generate this plasmid the PVY fragment from Cla pBC.PVY was cloned as a Xhol/EcoRl fragment into xhol/EcoRl-digested p35S.SCBV.O.SCBV.YVP.
  • Plasmid pART7.35S.PVYx3.SCBV.YVPx3 (FIG. 66) was designed to co-express sense and antisense repeats of PVY in transgenic plants. to generate this plasmid, the intermediate pART7.35S.O.SCBV.YVPx3 was constructed by cloning the triple direct PVY repeat from ClapBC.PVYx3 as a Clal/Accl fragment into Cla-digested p35S.SCBV.cass and isolating a plasmid with an antisense orientation.
  • Plasmid pART7.PVYx3.LNYV.YVPx3 (FIG. 67) was designed to express triple repeats of PVY sequences as an interrupted palindrome. To generate this plasmid an intermediate, pART7x3.PVY.LNYV.YV was constructed by cloning a PVY.LNYV.YVP fragment from pBC.PVY.LNYV.YVP as an Accl/Clal fragment into the plasmid pART7.PVYx2.
  • pART7.35S.PVYx3.LNYV.YVPx3 was made by cloning an additional PVY direct repeat from pBC.PVYx2 as an Accl/Clal fragment into Clal digested pART7x3.PVY.LNYV.YVP. Sequences from pART7.35S.PVYx3.LNYV.YVPx3, including the 35S promoter, all PVY sequences and the OCS terminator were excised as a Noti fragment and cloned into Noti-digested pART27, a plasmid with an appropriate orientation was chosen and designated pART27.35S.PVYx3.LNYV.
  • Plasmid pART7.35S.PVY multi (FIG. 68) was designed to express higher order direct repeats of regions of PVY sequences in transgenic plants. Higher order direct repeats of a 72 bp of the PVY Nia region from PVY were prepared by annealing two partially complementary oligonucleotides as follows: (SEQ ID NO:15) 5′-TAATGAGGATGATGTCCCTACCTTTAATTGGCAGAAATTTCTGTGGA AAGACAGGGAAATCTTTCGGCATTT-3′; and
  • oligonucleotides were phosphorylated with T4 pofynucleotide kinase, heated and cooled slowly to permit self-annealing, ligated with T4 DNA ligase, end-filled with Klenow polymerase and cloned into pCR2.1 (Invitrogen). Plasmids containing multiple repeats were isolated and sequences were cloned as EcoRi fragments in a sense orientation into EcoRl-digested pART7, to create the intermediate pART7.PVY multi.
  • pART27.PVY multi the 35S promoter, PVY sequences and the OCS terminator were excised as a Noti fragment and cloned into Notl-digested pART27.
  • a plasmid with an appropriate insert orientation was isolated and designated pART27.PVY multi.
  • Viral immune lines are created by expressing viral sequences in stably transformed cell lines.
  • lytic viruses are used for this approach since cell lysis provides very simple screens and also offer the ability to directly select for potentially rare transformation events which might create viral immunity.
  • Sub-genomic fragments derived from a simple single stranded RNA virus (Bovine enterovirus —BEV) or a complex double stranded DNA virus, Herpes Simplex Virus I (HSV I) are cloned into a suitable vector and expressed in transformed cells.
  • Mammalian cell lines are transformed with genetic constructs designed to express viral sequences driven by the strong cytomegalovirus (CMV-lE) promoter. Sequences utilised include specific viral replicase genes. Random “shotgun” libraries comprising representative viral gene sequences, may also be used and the introduced dispersed nucleic acid molecule, to target the expression of virus sequences.
  • CMV-lE cytomegalovirus
  • Resistant cell lines are supportive of the ability of the introduced nucleotide sequences to inactivate viral gene expression in a mammalian system.
  • resistant lines obtained from such experiments are used to more precisely define molecular and biochemical characteristics of the modulation which is observed.
  • Nicotiana tabaccum (cultivar W38) were transformed with these Agrobacterium strains using standard procedures. Putative transformed shoots were excised and rooted on media containing kanamycin. Under these conditions we have consistently observed that only transgenic shoots will root on kanamycin plates. Rooted shoots were transferred to soil and allowed to establish. After two to three weeks, vigorous plants with at least three sets of leaves were chosen and infected With PVY.
  • Viral inoculum was prepared from W38 tobacco previously infected with the virus, approximately 2 g of leaf material, showing obvious viral symptoms were ground with carbarundum in 10 ml of 100mM Na phosphate buffer (pH 7.5). the inoculum was diluted to 200 ml with additional Na phosphate buffer. Two leaves from each transgenic plant were sprinkled with carbarundum, then 0.4 ml of inoculum was applied to each leaf and leaves rubbed fairly vigorously with fingers. Using this procedure 100% of non-transgenic control plants were infected with PVY.
  • PVY-D an Australian PVY isolate
  • Transgenic lines were described as resistant if they showed reduced viral symptoms, which manifests as a reduction in the leaf are showing chlorotic lesions. Resistance ranges from very strong resistance where only a few viral lesions are observed on a plant to weak resistance which manifects as reduced symptoms on leaves that develop late in plant growth.
  • inverted and/or direct repeat sequences modulate expression of the virus target gene in the transgenic plant.
  • porcine PK2 cells were transformed with the relevant constructs.
  • PK2 cells constitutively express Galt enzyme, the activity of which results in the addition of a variety of a:-1,3-galactosyl groups to a range of proteins expressed on the cell surface of these cells.
  • Cells were transformed using lipofectin and stably transformed lines were selected using genetecin.
  • HBSS/Hepes Hank's buffered saline solution with 20 mM Hepes, pH7.4
  • IB4-biotin 10 ug/ml IB4-biotin (Sigma) in HBSS/Hepes for 45 mins at 4° C.
  • Cells were washed in HBSS/Hepes, probed with a 1:200 dilution of ExtrAvidin-FITC (Sigma) in HBSSYHepes for 45 mins at 4° C. at and rinsed in cold HBSS/Hepes prior to FACS sorting.
  • RNA was purified from the indicated cell lines using an RNeasy Mini Kit according to the supplier's protocol (Qiagen). To prepare cDNA, this RNA was reverse-transcribed using Omniscript Reverse Transcriptase (Qiagen). Two micrograms of total RNA was reverse-transcribed using 1iM oligo dT (Sigma) as a primer in a 20 ⁇ l reaction according to the supplier's protocol (Qiagen).
  • PCR amplification conditions involved an initial activation step at 950C for 15 min, followed by 35 amplification cycles of 94° C for 30 sec, 60° C. for 30 sec and 72° C. for 60 sec. with a final elongation step at 72° C. for 4 min.
  • PCR products to be cloned were usually purified using a QlAquick PCR Purification Kit (Qiagen); in instances where multiple fragments were generated by PCR, the fragment of the correct size was purified from agarose gels using a QlAquick Gel Purification Kit (Qiagen) according to the supplier's protocol.
  • Qiagen QlAquick PCR Purification Kit
  • Amplification products were then cloned into pCR (registered trademark) 2.1-TOPO (Invitrogen) according to the supplier's protocol.
  • insert fragments were excised from intermediate vectors using restriction enzymes according to the supplier's protocols (Roche) and fragments purified from agarose gels using QlAquick Gel Purification Kits (Qiagen) according to the supplier's protocol.
  • Vectors were usually prepared by restriction digestion and treated with Micromp Alkaline Phosphatase according to the supplier's protocol (Amersham).
  • Vector and inserts were ligated using T4 DNA ligase according to the supplier's protocols (Roche) and transformed into competent Escherichia coli strain DH5o using standard procedures (Sambrook, Fritsch et al. 1989).
  • Plasmid pEGFP-N1 (Clontech) contains the CMV IE promoter operably connected to an open reading frame encoding a red-shifted variant of the wild-type GFP which has been optimized for brighter fluorescence.
  • the specific GFP variant encoded by pEGFP-N1 has been disclosed by (Cormack, Valdivia et al. 1996).
  • Plasmid pEGFP-N1 contains a multiple cloning site comprising BgIll and BamHl sites and many other restriction endonuclease cleavage sites, located between the CMV IE promoter and the EGFP open reading frame, The plasmid pEGFP-N1 will express the EGFP protein in mammalian cells.
  • the plasmid further comprises an SV40 polyadenylation signal downstream of the EGFP open reading frame to direct proper processing of the 3′-end of mRNA transcribed from the CMV IE promoter sequence (SV40 pA).
  • the plasmid further comprises the SV40 origin of replication functional in animal cells; the neomycin-resistance gene comprising the SV40 E (early) promoter operably connected to the neomycin/kanamycin-resistance gene derived from Tn5 and the HSV thymidine kinase polyadenylation signal, for selection of transformed cells on kanamycin, neomycin or geneticin; the pUC19 origin of replication which is functional in bacterial cells and the f1 origin of replication for single-stranded DNA production.
  • the neomycin-resistance gene comprising the SV40 E (early) promoter operably connected to the neomycin/kanamycin-resistance gene derived from Tn5 and the HSV thymidine kinase polyadenylation signal, for selection of transformed cells on kanamycin, neomycin or geneticin
  • the pUC19 origin of replication which is functional in bacterial cells and
  • Plasmid pBluescript 11 SK+ (Stratagene) comprises the lacZ promoter sequence and lacz-a transcription terminator, with multiple restriction endonuclease cloning sites located there between. Plasmid pBluescript ll SK + is designed to clone nucleic acid fragments by virtue of the multiple restriction endonuclease cloning sites.
  • the plasmid further comprises the ColEl and f1 origins of replication and the ampicillin-resistance gene ( ⁇ -lactamase).
  • Plasmid pCR (registered trademark) 2.1 (Invitrogen ) is a T-tailed vector comprising the lacZ promoter sequence and lacZ- ⁇ transcription terminator, with a cloning site for the insertion of structural gene sequences there between. Plasmid pCR (registered trademark) 2.1 is designed to clone nucleic acid fragments by virtue of the A-overhang frequently synthesized by Taq polymerase during the polymerase chain reaction. The plasmid further comprises the ColEl and f1 origins of replication and kanamycin-resistance and ampicillin-resistance genes.
  • Plasmid pCR (registered trademark) 2.1-TOPO is a T-tailed vector comprising the lacz promoter sequence and lacZ- ⁇ transcription terminator, with multiple restriction endonuclease cloning sites located there between. Plasmid pCR (registered trademark) 2.1-TOPO is provided with covalently bound topoisomerase I enzyme for fast cloning. The plasmid further comprises the ColE] and f1 origins of replication and the kanamycin and ampicillin-resistance genes.
  • Plasmid TOPO-BGI2 comprises the human P-globin intron number 2 (BGI2) placed in the multiple cloning region of plasmid pCR (registered trademark) 2.1-TOPO. To produce this prasmid, the human P-globin intron number 2 (BGI2) was amplified from human genomic DNA using the amplification primers:
  • BGI2 is a functional intron sequence that is capable of being post-transcriptionally cleaved from RNA transcripts containing it in mammalian cells.
  • Plasmid pCMV.cass is an expression cassette for driving expression of a structural gene sequence under control of the CMV-lE promoter sequence.
  • Plasmid pCMV.cass was derived from pEGFP-N1 by deletion of the EGFP open reading frame as follows: Plasmid pEGFP-N1 was digested with PinAl and Nod, blunt-ended using Pful DNA polymerase and then religated. Structural gene sequences are cloned into pCMV.cass using the multiple cloning site, which is identical to the multiple cloning site of pEGFP-N1, except it lacks the PinAl site.
  • RNAs transcribed from the CMV promoter will include the human P-globin intron 2 sequences; these intron sequences will presumably be excised from transcripts as part of the normal intron processing machinery, since the intron sequences include both the splice donor and splice acceptor sequences necessary for normal intron processing.
  • PK-1 cells (derived from porcine kidney epithelial cells) were grown as adherent monolayers using DMEM supplemented with 10% v/v FBS.
  • Plasmid pBluescript.EGFP comprises the EGFP open reading frame derived from plasmid pEGFP-Nl placed in the multiple cloning region of plasmid pBluescript ll SK + (Stratagene). To produce this plasmid, the EGFP open reading frame was excised from pEGFP-Nl by restriction endonuclease digestion using the enzymes Nod and Xhol and ligated into Nodl/Xhol-digested pBluescript ll SK;.
  • Plasmid pCMV.EGFP is capable of expressing the entire EGFP open reading frame under the control of CMV-lE promoter sequence.
  • the EGFP sequence from pBluescript.EGFP was sub-cloned in the sense orientation as a BamHl-to-Sad fragment into Bgll/Sacl-digested pCMV.cass.
  • Transformations were performed in 6-well tissue culture vessels (Nunc). Individual wells were seeded with 4 ⁇ 10 4 PK-1 cells in 2 ml of DMEM, 10% v/v FBS and incubated at 37° C. in 5% v/v CO 2 until the monolayer was 60-90% confluent, typically 16 to 24 hr.
  • tissue growth medium was removed from each well and the monolayer therein was washed with 1 ml of 1 x PBS (Sigma).
  • the monolayers were overlayed with 1 ml of the plasmid DNA/GenePORTER2 (trademark) conjugate for each well and incubated at 37° C. in 5% V/V CO 2 for 4.5 hr.
  • OPTT-MEM-I registered trademark
  • 20% vIv FBS was added to each well and the vessel incubated for a further 24 hr, at which time the monolayers were washed with 1 x PBS and medium was replaced with 2 ml of fresh DMEM including 10% v/v FBS.
  • Cells transformed with pCMV.EGFP were examined after 24-48 hr for transient EGFP expression using fluorescence microscopy at a wavelength of 500-550 nm.
  • a number of parental cell lines were transformed with pCMV.EGFP. Following continuous culture, in many of these lines GFP expression was either extremely low or completely undetectable as listed in Table 3 and shown in FIG. 42. TABLE 3 Number of cell lines Number of cloned lines with extremely low or Parental Cell line examined undetectable GFP PK-1 (pig) 59 2 MM96L (human) 12 4 B16 (mouse) 12 10 MDAMB468 (human) 11 1
  • CRIB-1 cells (derived from bovine kidney epithelial cells) were grown as adherent monolayers using DMEM supplemented with 10% v/v Donor Calf Serum (DCS; Life Technologies), as described in Example 1.
  • Bovine enterovirus (BEV) RNA polymerase coding region was amplified from a full-length cDNA clone encoding same, using primers: BEV-1 CGG CAG ATC CTA ACA ATG GCA [SEQ ID NO:19] GGA CAA ATC GAG TAC ATC
  • Primer BEV-1 comprises a Bglll restriction endonuclease site at positions 4-9, inclusive, and an ATG start site at positions 16-18, inclusive.
  • Primer BEV-3 comprises a BamHl restriction enzyme site at positions 5-10, inclusive, and the complement of a TAA translation stop signal at positions 11-13, inclusive.
  • the amplified fragment was cloned into pCR2.1 to produce plasmid pCR.BEV2.
  • Plasmid pBS.PFGE contains the,EGFP coding sequences,from pEGFP-N1 cloned in antisense orientation into the polylinker of pBluescript 11 SK+. To generate this plasmid, the EGFP coding sequences from pEGFP-Nl was cloned as a Notl-to-Sacf fragment into Notg/Sacl-digested pBluescript 11 SK+.
  • Plasmid pCMV.BEV2.BGI2.2VEB contains an inverted repeat or palindrome of the BEV polymerase coding region that is interrupted by the insertion of the human ⁇ -globin intron 2 sequence therein.
  • Plasmid pCMV.BEV2.BG[2.2VEB was constructed in successive steps: (i) the BEV2 sequence from plasmid pCR.BEV2 was sub-cloned in the sense orientation as a Bgfll-to-BamHI fragment into Bg/ll-digested pCMV.BGI2.cass to make plasmid pCMV.BEV2.BGI2, and (ii) the BEV2 sequence from plasmid pCR.BEV2 was sub-cloned in the antisense orientation as a Bg/ll-to-BamHl fragment into BamHl-digested pCMV.BEV2.BGI2 to make plasmid pCMV.BEV2.BGI2.2VEB.
  • Plasmid pCMV.BEV.EGFP.VEB contains an inverted repeat or palindrome of the BEV polymerase coding region that is interupted by EGFP coding sequences which act as a stuffer fragment
  • the EGFP coding sequence from pBS.PFGE was isolated as an EcoRl fragment and cloned into EcoRl-digested pCMV.cass in the sense orientation relative to the CMV promoter to generate pCMV.EGFP.cass.
  • Plasmid pCMV.BEV.EGFP.VEB was constructed in successive steps: (i) the BEV polymerase sequence from plasmid pCR.BEV2 was sub-cloned in the sense orientation as a 13gIl-to-BamHI fragment into Bg/ll-digested pCMV.EGFP.cass to make plasmid pCMV.BEV.EGFP, and (ii) the BEV polymerase sequence from plasmid pCR.BEV2 was sub-cloned in the antisense orientation as a Bg/ll-to-BamHl fragment into BamHl-digested pCMV.BEV.EGFP to make plasmid pCMV. BEV.EGFP.VEB.
  • Transformations were performed in 6-well tissue culture vessels. Individual wells were seeded with 2 ⁇ 10 5 CRIB-1 cells in 2 ml of DMEM, 10% v/v DCS and incubated at 37° C. in 5% v/v CO 2 until the monolayer was 60-90% confluent, typically 16-24 hr.
  • Solution A For each transfection, 1 ⁇ g of DNA (pCMV.BEV2.BGI2.2VEB or pCMV.EGFP) was diluted into 100 ⁇ l of OPTI-MEM-l (registered trademark) and;
  • Solution B For each transfection, 10 ⁇ l of LIPOFECTAMINE (trademark) Reagent (Life Technologies) was diluted into 100 ⁇ l OPTI-MEM-1 (registered trademark).
  • Transfection mixture was then removed and the CRIB-1 monolayers overlaid with 2 ml of DMEM, 10% v/v DCS.
  • Cells were incubated at 37° C. in 5% V/V CO 2 for approximately 48 hr. To select for stable transformants, the medium was replaced every 72 hr with 4 ml of DMEM, 10% v/v DOCS, 0.6 mg/ml geneticin.
  • the BEV isolate used in these experiments was a cloned isolate, K2577.
  • cells were infected with 5 ⁇ l of viral stock per well and the virus allowed to replicate for 48 hr, as described below, Culture medium was harvested at this time and transferred to a screw-capped tube. Dead cells and debris were removed by centrifugation at 3,500 rpm for 15 min at 4° C. in a Sigma 3K18 centrifuge. The supernatant was decanted into a fresh tube and centrifuged at 20,000 rpm for 30 min at 40° C. in a Beckman J2-M1 centrifuge to remove remaining debris. The supernatant was decanted and this new BEV stock titred as described below and stored at 4° C.
  • BEV was diluted in serum-free DMEM at dilutions of 10 ⁇ 1 to 10 ⁇ 9.
  • Medium was aspirated from the CRIB-1 monolayers and the cells overlaid with 2 ml of 1 x PBS and the vessels rocked gently to wash the monolayer.
  • PBS was aspirated from the monolayer and the wash repeated.
  • the agar overlay was allowed to set and the plates incubated (inverted) at 37° C. in 5% v/v CO 2 for 18-24 hr. Following incubation, each well was overlaid with 3 ml of Neutral Red Agar (1.7 ml Neutral Red Solution (Life Technologies) in 100 ml Nutrient Agar). The overlay was allowed to set and the plates incubated (inverted) in the dark at 37° C. in 5% V/V CO 2 for 18-24 hr. Plaques were counted to determine the titre of the BEV viral stock.
  • BEV was diluted in serum-free DMEM at dilutions of 10 ⁇ 1 to 10 ⁇ 9.
  • the medium was aspirated from the CRIB-1 monolayers and the monolayers overlaid with 800 ⁇ l of 1 x PBS and washed by gently rocking the tissue culture vessel. PBS was aspirated from the monolayers and the wash repeated.
  • BEV virus was diluted in serum-free DMEM at an appropriate dilution.
  • the BEV viral stock was diluted to 10x and 0.1x the working dilution (typically 10 ⁇ 4 to 10 ⁇ 6 pfu).
  • FIG. 44 shows micrographs comparing CRIB-1 and CRIB-i BG]2 # 19(tol) cells before and 48 hr after BEV infection.
  • B16 cells derived from murine melanoma (ATCC CRL-6322) were grown as adherent monolayers in RPMI 1640 supplemented with 10% v/v FBS, as described in Example 1.
  • TYR-F GTT TCC AGA TCT CTG ATG GC [SEQ ID NO:21]
  • TYR-R AGT CCA CTC TGG ATC CTA GG [SEQ ID NO:22],
  • PCR amplification was performed using HotStarTaq DNA polymerase according to the supplier's protocol (Qiagen). PCR amplification conditions involved an initial activation step at 95° C. for 15 mins, followed by 35 amplification cycles of 94° C. for 30 sec, 55° C. for 30 sec and 72° C. for 60 sec, with a final elongation step at 72° C. for 4 min.
  • PCR amplified region of tyrosinase was column-purified (PCR purification column, Qiagen) and then cloned into pCR (registered trademark) 2.1-TOPO according to the supplier's instructions (Invitrogen) to make plasmid TOPO.TYR.
  • Plasmid pCMV.TYR.BGI2.RYT contains an inverted repeat, or palindrome, of a region of the murine tyrosinase gene that is interrupted by the insertion of the human ⁇ -globin intron 2 (BGI2) sequence therein.
  • Plasmid pCMV.TYR.BGI2.RYT was constructed in successive steps: (i) the TYR sequence from plasmid TOPO.TYR was sub-cloned in the sense orientation as a Bg/ll-to-BamHl fragment into Bg/ll-digested pCMV.BGI2 to make plasmid pCMV.TYR.BG)2, and (ii) the TYR sequence from plasmid TOPO.TYR was sub-cloned in the antisense orientation as a Bg/ll-to-BamHl fragment into BamHl-digested pCMV.TYR.BGI2 to make plasmid pCMV.TYR.BGI2.RYT.
  • Plasmid pCMV.TYR contains a single copy of mouse tyrosinase CDNA sequence, expression of which is driven by the CMV promoter. Plasmid pCMV.TYR was constructed by cloning the TYR sequence from plasmid TOPO.TYR as a BamHl-to-Bg/ll fragment into BamHl-digested pCMV.cass and selecting plasmids containing the TYR sequence in a sense orientation relative to the CMV promoter.
  • Plasmid pCMV.TYR.TYR contains a direct repeat of the mouse tyrosinase cDNA sequence, expression of which is driven by the CMV promoter. Plasmid pCMV.TYR.TYR was constructed by cloning the TYR sequence from plasmid TOPO.TYR as a BarnHl-to-BgAl fragment into BamHi-digested pCMV.TYR and selecting plasmids containing the second TYR sequence in a sense orientation relative to the CMV promoter.
  • Tyrosinase is the major enzyme controlling pigmentation in mammals. If the gene is inactivated, melanin will no longer be produced by the pigmented B16 melanoma cells. This is essentially the same process that occurs in albino animals.
  • Transformations were performed in 6-well tissue culture vessels. Individual wells were seeded with 1 ⁇ 10 5 cells in 2 ml of RPMI 1640, 10% v/V FBS and incubated at 37° C. in 5% V/V CO 2 until the monolayer was 60-90% confluent, typically 16-24 hr.
  • Cell populations to be stained were resuspended at a concentration of 500,000 cells per ml in RPMI 1640 medium. Volumes of 200 ⁇ l were dropped onto surface-sterilized microscope slides and slides were incubated at 37° C. in a humidified atmosphere in 100 mm TC dishes until cells had adhered firmly. The medium was removed and cells were fixed by air drying on a heating block at 37° C. for 30 min then post-fixed with 4% w/v paratormaldehyde (Sigma) in PBS for 1 hr. Fixed cells were hydrated by dipping in 96% v/v ethanol in distilled water, 70% v/v ethanol, 50% v/v ethanol then distilled water.
  • Slides with adherent cells were left for 1 hr in a ferrous sulfate solution (2.5% w/v ferrous sulfate in water) then rinsed in four changes of distilled water , 1 min each. Slides were left for 30 min in a solution of potassium ferricyanide (1% w/v potassium ferricyanide in 10% v/v acetic acid in distilled water). Slides were dipped in 1% v/v acetic acid (15 dips) then dipped in distilled water (15 dips).
  • Tyrosinase catalyzes the first two steps of melanin synthesis: the hydroxylation of tyrosine to dopa (dihydroxyphenylalanine) and the oxidation of dopa to dopaquinone. Tyrosinase can be measured as its dopa oxidase activity.
  • This assay uses Besthorn's hydrazone (3-methyl-2-benzothiazolinonehydrazone hydrochloride, MBTH) to trap dopaquinone formed by the oxidation of L-dopa. Presence of a low concentration of N,N′-dimethylformamide in the assay mixture renders the MBTH soluble and the method can be used over a range of pH values.
  • MBTH reacts with dopaquinone by a Michael addition reaction and forms a dark pink product whose presence is monitored using a spectrophotometer or plate reader. It is assumed that the reaction of the MBTH with dopaquinone is very rapid relative to the enzyme-catalyzed oxidation of L-dopa. The rate of production of the pink pigment can be used as a quantitative measure of enzyme (Winder and Harris 1991; Dutkiewicz, Albert et al, 2000).
  • B16 cells and transformed B16 cell lines were plated into individual wells of a 96-well plate in triplicate. Constant numbers of cells (25,000) were transferred into individual wells and cells were incubated overnight. Tyrosinase assays were performed as described below after either 24 or 48 hr incubation.
  • Tyrosinase activity was assayed by adding 190 ul freshly-prepared assay buffer (6.3mM MBTH, 1.mM L-dopa, 4% v/v N,N′-dimethylformamide in 48mM sodium phosphate buffer (pH 7.1)) to each well. Colour formation was monitored at 505 nm in a Tecan plate reader and data collected using X/Scan Software. Readings were taken at constant time intervals and reactions monitored at room temperature, typically 22° C. Results were calculated as the average of enzyme activities as measured for the triplicate samples. Data were analyzed and tyrosinase activity estimated at early time-points when product formation was linear, typically between 2 and 12 min.
  • HER-2 (also designated neu and erbB-2) encodes a 185 kDa transmembrane receptor tyrosine kinase that is constitutively activated at low levels and displays potent oncogenic activity when over-expressed.
  • HER-2 protein over-expression occurs in about 30% of invasive human breast cancers. The biological function of HER-2 is not well understood. It shares a common structural organisation with other members of the epidermal growth factor receptor family and may participate in similar signal transduction pathways leading to changes in cytoskeleton reorganisation, cell motility, protease expression and cell adhesion. Over-expression of HEFR-2 in breast cancer cells leads to increased tumorigenicity, invasiveness and metastatic potential (Slamon, Clark et al. 1987).
  • Human MDA-MB-468 cells were cultured in RPMI 1640 supplemented with 10% v/v FBS. Cells were passaged twice a week by treating with trypsin to release cells and transferring a proportion of the culture to fresh medium, as described in Example 1.
  • a region of the human HER-2 gene was amplified by PCR using human cDNA as a template.
  • the cDNA was prepared from total RNA isolated from a human breast tumour line, SK-BR-3. Total RNA was purified as described in Example 8.
  • Human HER-2 sequences were amplified using the primers:
  • H3 GTC GAC TGT GTT CCA TCC TCT GOCT GTC AC [SEQ ID NO:241].
  • Plasmid pCMV.HER2.BGl2.2REH contains an inverted repeat or palindrome of the HER-2 coding region that is interrupted by the insertion of the human ⁇ -globin intron 2 (BGI2) sequence therein.
  • Plasmid pCMV.HER2.BGl2.2REH was constructed in successive steps: (i) the HER-2 sequence from plasmid TOPO.HER2 was sub-cloned in the sense orientation as a SaAlXhol fragment into Sat-digested pCMV.BGI2.cass (Example 6) to make plasmid pCMV.HER2.BGI2, and (ii) the HER2 sequence from plasmid TOPO.HER2 was sub-cloned in the antisense orientation as a SaAlXhol fragment into Xhol-digested pCMV.HER2.BGI2 to make plasmid pCMV.HER2.BGI2.2REH.
  • Transformations were performed in 6-well tissue culture vessels. Individual wells were seeded with 4 ⁇ 10 5 MDA-MB-468 cells in 2 ml of RPMI 1640 medium, 10% v/v FIBS and incubated at 37° C. in 5% v/v CO 2 until the monolayer was 60-90% confluent, typically 16-24 hr.
  • MDA-MB-468 cells over-express HER-2 and PTGS of the gene in geneticin-selected clones derived from this cell line were tested by immunofluorescence labelling of clones (see Example 1) with a primary murine monoclonal antibody directed against the extracellular domain of HER-2 protein.
  • the primary antibody was a mouse Anti-erbB2 monoclonal antibody (Transduction Laboratories, Cat. No. E19420, an IgG2b isotype) used at 11400 dilution; the secondary antibody was Alexa Fluor 488 goat anti-mouse IgG (H+L) conjugate (Molecular Probes, Cat. No. A-11001) used at 11100 dilution.
  • MDA-MB-468 cells parental and transformed lines
  • Selected clones and control MDA-MB-468 cells were grown overnight to near-confluence on 100 mm TC plates (10 7 cells). Cells in plates were first washed with buffer containing phosphatase inhibitors (50mM Tris-HCI, pH 6.8, 1mM Na 4 P 2 O 7 , 10mM NaF, 20 ⁇ M Na 2 MoO 4 , 1mM Na 3 VO 4 ), and then scraped from the plate in 600 ⁇ l of lysis buffer (50mM Tris-HCI, pH 6.8, 1mM Na 4 P 2 O 7 , 10mM NaF, 20 ⁇ M Na 2 MoO 4 , 1mM Na 3 VO 4 , 2% w/v SDS) which had been heated to 100° C. Suspensions were incubated in screw-capped tubes at 100° C. for 15 min. Tubes with lysed cells were centrifuged at 13,000 rpm for 10 min and supernatant extracts were removed and stored at ⁇ 20° C.
  • Membranes were rinsed in TBST buffer (10mM Tris-HCI, pH 8.0, 150mM NaCI, 0.05% v/v Tween 20) then blocked in a dish in TBST with 5% w/v skim milk powder plus phosphatase inhibitors (1mM Na 4 P 2 O 7 , 10mM NaF, 20,uM Na 2 MoO 4 , 1 mM Na 3 VO 4 ). Membranes were incubated in a small volume in TBST with 2.5% w/v skim milk powder plus phosphatase inhibitors containing a mouse monoclonal antibody against the ECD of HER-2 (Transduction Laboratories, NeoMarkers) diluted 1:4000.
  • TBST buffer 10mM Tris-HCI, pH 8.0, 150mM NaCI, 0.05% v/v Tween 20
  • phosphatase inhibitors 1mM Na 4 P 2 O 7 , 10mM NaF, 20,uM Na 2 MoO 4 , 1
  • Membranes were washed three times for 10 min in TBST with 2.5% w/v skim milk powder plus phosphatase inhibitors. Membranes were incubated in a small volume in TBST with 2.5% w/v skim milk powder plus phosphatase inhibitors containing the horseradish peroxidase-conjugated secondary antibody diluted 1:1000. Membranes were washed three times for 10 min in TBST with 2.5% w/v skim milk powder plus phosphatase inhibitors.
  • HER-2 protein was detected using the ECL luminol-based system (Amersham), according to manufacturer's instructions.
  • ECL luminol-based system Amersham
  • Several cell lines transformed with pCMV.HER2.BGI2.2REH showed greatly reduced or no detectable HER-2 protein.
  • BIO.2 calls (Immunex) derived from murine fibrosarcoma and Pam 212 cells (Auckland Medical School) derived from sponstaneously transformed murine epidermal keratinocytes were grown as adherent monolayers in cDMEM (DMEM with 0.77mM asparagine, 160 ⁇ M penicillin G, 70 ⁇ M dihydrostreptomycin sulfate) supplemented with 5% v/v FBS (B10.2) or 5% v/v equine serum (Pam 212), as described in Example 1, above.
  • cDMEM DMEM with 0.77mM asparagine, 160 ⁇ M penicillin G, 70 ⁇ M dihydrostreptomycin sulfate
  • 5% v/v FBS B10.2
  • 5% v/v equine serum (Pam 212), as described in Example 1, above.
  • Y1 AGA TCT GCA GCA GAC CGT AAC CAT TAT AGG [SEQ ID NO:25].
  • Y4 GGA TCC ACC TTT ATT AAC AGG TGC TTG CAG (SEQ ID NO:261.
  • PCR amplification was performed using HotStarTaq DNA polymerase according to the supplier's protocol (Qiagen). PCR amplification conditions involved an initial activation step at 95° C. for 15 min, followed by 35 amplification cycles of 94° C. for 30 sec, 55° C. for 30 sec and 72° C. for 60 sec, with a final elongation step at 72° C. for 4 min.
  • PCR-amplified region of YB-1 was column-purified (PCR purification column, Qiagen) and then cloned into pCR (registered trademark) 2.1-TOPO (Invitrogen) according to the supplier's instructions, to make plasmid TOPO.YB-1.
  • P4 GGA TCC CAG GCC CCA CTT TCT TGA CCA TTG [SEQ ID NO;28].
  • PCR amplification was performed using HotStarTaq DNA polymerase according to the supplier's protocol (Qiagen). PCR amplification conditions involved an initial activation step at 95° C. for 15 min, followed by 35 amplification cycles of 94° C. for 30 sec, 55° C. for 30 sec and 720C for 60 sec, with a final elongation step at 72°C. for 4 min.
  • PCR-amplified region of p53 was column-purified (PCR purification column, Qiagen) and then cloned into pCR (registered trademark) 2.1-TOPO (Invitrogen) according to the manufacturer's instructions, to make plasmid TOPO.p53.
  • the murine YB-1 sequence from TOPO.YB-1 was isolated as a Bg/ll-to-BamHl fragment and cloned into the BamHl site of TOPO.p53.
  • a clone in which the YB-1 insert was oriented in the same sense as the p53 sequence was selected and designated TOPO.YB1.p53.
  • Plasmid pCMV.YB1 .BGI2.1 BY is capable of transcribing a region of the murine YB-1 gene as an inverted repeat or palindrome that is interrupted by the human ⁇ -globin intron 2 (BGI2) sequence therein.
  • Plasmid pCMV.YBI.BGI2.1BY was constructed in successive steps: (i) the YB-1 sequence from plasmid TOPO.YB-1 was sub-cloned in the sense orientation as a Bg/ll-to-BamHl fragment into Bg/ll-digested pCMV.BGI2 to make plasmid pCMV.YBI.BGI2, and (ii) the YB-1 sequence from plasmid TOPO.YB-1 was sub-cloned in the antisense orientation as a Bg/ll-to-BamHl fragment into BamHl-digested pCMV.YB1.BGI2 to make plasmid pCMV.YB1.BGI2.1 BY.
  • Plasmid pCMV.YB1 p53.BGI2.35p.lBY is capable of expressing fused regions of the murine YB-1 and p53 genes as an inverted repeat or palindrome that is interrupted by the human ⁇ -globin intron 2 (BGI2) sequence therein.
  • Plasmid pCMV.YBi.p53.BG[2.35p.1BY was constructed in successive steps: (i) the YB-1.p53 fusion sequence from plasmid TOPO.YB1.p53 was sub-cloned in the sense orientation as a Bg/ll-to-BamHl fragment into Bg/ll-digested pCMV.BGI2 to make plasmid pCMV.YB1.p53.BGI2, and (ii) the YB-i.p53 fusion sequence from plasmid TOPO.YBI.p53 was sub-cloned in the antisense orientation as a Bg/ll-to-BamHl fragment into BamHi-digested pCMV.YB1.p53,BGI2 to make plasmid pCMV.YB1 .p53.BGI2.35p.1 BY.
  • YB-1 (Y-box DNA/RNA-binding factor 1) is a transcription factor that binds, inter alia, to the promoter region of the p53 gene and in so doing represses its expression.
  • YB-i a transcription factor that binds, inter alia, to the promoter region of the p53 gene and in so doing represses its expression.
  • the murine cell lines Bi0.2 and Pam 212 are two such tumorigenic cell lines with normal p53 expression. The expected phenotype for co-suppression of YB-1 in these two cell lines is apoptosis.
  • Transformations with pCMV.YB1.BGI2.1BY were performed in 6-well tissue culture vessels. Individual wells were seeded with 3.5 ⁇ 10 4 cells (B10.2 or Pam 212) in 3 ml of cDMEM, 5% v/v FBS (B10.2) or equine serum (Pam 212) and incubated at 37° C. in 5% v/v CO 2 for 24 hr prior to transfection.
  • Mix B 1 ⁇ l (400 ng) of pCMV.YBI.BGI2.1BY DNA in 100 ⁇ l of OPTI-MEM I (registered trademark) medium.
  • Live and dead cell numbers were determined by trypan blue staining (0.2%) and counting in quadruplicate on a haemocytometer slide. Results are presented in FIG. 75.
  • Transformations with pCMV.YBI .p53.BGI2.35p.1 BY were performed in 6-well tissue culture vessels. Individual wells were seeded with 3.5 ⁇ 10 4 cells (B10.2 or Pam 212) in 3 ml of cDM EM, 5% v/v FBS (B10.2) or equine serum (Pam 212) and incubated at 37° C. in 5% v/V CO 2 for 24 hr prior to transfection.
  • Live and dead cell numbers were determined by trypan blue staining (0.2%) and counting in quadruplicate on a haemocytometer slide. Results are presented in FIG. 75.
  • Transformations with pCMV.EGFP were performed in 6-well tissue culture vessels. Individual wells were seeded with 3.5 ⁇ 10 4 cells (B1O.2 or Pam 212) in 3 ml of cDMEM, 5% v/v FBS (B1O.2) or equine serum (Pam 212) and incubated at 37° C. in 5% V/V CO 2 for 24 hr prior to transfection.
  • Mix B 1 ⁇ l (400 ng) of pCMV.EGFP DNA in 100 ⁇ l of OPTI-MEM I (registered trademark) medium.
  • Live and dead cell numbers were determined by trypan blue staining (0.2%) and counting in quadruplicate on a haemocytometer slide. Results are presented in FIG. 75.
  • YB1 decoy GAA CCT GAA TTT GGA TGC AGT TCC AGA C CTT GGA CTT AAA CCT ACG TCA AGG TCT G [SEQ ID NO:30]
  • YB1 control GCG GAT AAC AAT TTC ACA CAG G CGC CTA TTG TTA AAG TGT GTC C
  • Transformations with YB1 decoy and a control (non-specific) oligonucleotide were performed in 24 well tissue culture vessels, Individual wells were seeded with 3.5 ⁇ 10 4 cells (610.2 or Pam 212) in 3 ml of cDMEM, 5% v/v FBS (B10.2) or equine serum (Pam 212) and incubated at 37° C., 5% VIV CO 2 for 24 hr prior to transfection.
  • Mix B 0.4 pi (40 pmol) of oligonucleotide (YB1 decoy or control) in 100 ⁇ l of OPTI-MEM I (registered trademark) medium.
  • a no-oligonucleotide (Lipofectin (trademark) only) control was also prepared.
  • FIG. 76 there is shown a Northem blot as explained in the description of FIG. 76 set out above.
  • This blot shows that clone #18 may be regarded as a “positive control”, in that there was no significant silencing.
  • clones #3 and #9 do show silencing as can be seen from the absence of a band in their respective lanes in the Northern blot of FIG. 76.
  • FIG. 77 graphs the results of Real-Time RT-PCR analysis of these cell lines, as explained in the description of FIG. 77 set out above. While it would be apparent to one skilled in the art the method used to generate these graphs, the method is set out in more detail in U.S. Provisional Patent application Ser. No. 60/316,308.
  • results of these graphs are tabulated for reference purposes in the table set out in FIG. 78.
  • the results for clone #18 show that the mRNA level for EGFP is set as the standard (1.000) relative to which other mRNA levels are measured. It can be seen that there is also significant mRNA present for GAPD (little under half the amount for EGFP). Under the heading “Transcription”, it can be seen that there is a measurable rate of mRNA transcription for EGFP and a lower rate of transcription measurable for GAPD.
  • the results for clone #3 are shown on the second line. Consistent with the results of FIG.
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