WO2005102041A2 - Crustacean expression vector - Google Patents
Crustacean expression vector Download PDFInfo
- Publication number
- WO2005102041A2 WO2005102041A2 PCT/US2005/012979 US2005012979W WO2005102041A2 WO 2005102041 A2 WO2005102041 A2 WO 2005102041A2 US 2005012979 W US2005012979 W US 2005012979W WO 2005102041 A2 WO2005102041 A2 WO 2005102041A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- shrimp
- promoter
- constract
- ires
- protein
- Prior art date
Links
- 241000238424 Crustacea Species 0.000 title claims abstract description 33
- 239000013604 expression vector Substances 0.000 title description 14
- 241000238557 Decapoda Species 0.000 claims abstract description 124
- 108020004684 Internal Ribosome Entry Sites Proteins 0.000 claims abstract description 64
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 62
- 108700026244 Open Reading Frames Proteins 0.000 claims abstract description 56
- 241000700605 Viruses Species 0.000 claims abstract description 54
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 50
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 49
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 49
- 238000000034 method Methods 0.000 claims abstract description 40
- 241000238631 Hexapoda Species 0.000 claims abstract description 31
- 206010033799 Paralysis Diseases 0.000 claims abstract description 7
- 108090000623 proteins and genes Proteins 0.000 claims description 99
- 210000004027 cell Anatomy 0.000 claims description 79
- 239000013598 vector Substances 0.000 claims description 75
- 241001265687 Taura syndrome virus Species 0.000 claims description 70
- 241000696962 White spot syndrome virus Species 0.000 claims description 42
- 241000439574 Decapod penstyldensovirus 1 Species 0.000 claims description 41
- 241000272443 Penelope Species 0.000 claims description 29
- 108091029795 Intergenic region Proteins 0.000 claims description 28
- 239000013612 plasmid Substances 0.000 claims description 27
- 230000003612 virological effect Effects 0.000 claims description 20
- 241001465754 Metazoa Species 0.000 claims description 19
- 230000014509 gene expression Effects 0.000 claims description 17
- 108091026898 Leader sequence (mRNA) Proteins 0.000 claims description 7
- 239000003550 marker Substances 0.000 claims description 7
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 claims description 6
- MJNIWUJSIGSWKK-UHFFFAOYSA-N Riboflavine 2',3',4',5'-tetrabutanoate Chemical compound CCCC(=O)OCC(OC(=O)CCC)C(OC(=O)CCC)C(OC(=O)CCC)CN1C2=CC(C)=C(C)C=C2N=C2C1=NC(=O)NC2=O MJNIWUJSIGSWKK-UHFFFAOYSA-N 0.000 claims description 5
- 230000010076 replication Effects 0.000 claims description 5
- 230000001105 regulatory effect Effects 0.000 claims description 4
- 230000001225 therapeutic effect Effects 0.000 claims description 4
- 210000004102 animal cell Anatomy 0.000 claims description 2
- 230000001681 protective effect Effects 0.000 claims 2
- 241000251468 Actinopterygii Species 0.000 abstract description 9
- 235000015170 shellfish Nutrition 0.000 abstract description 8
- 238000004519 manufacturing process Methods 0.000 abstract description 6
- 241000237852 Mollusca Species 0.000 abstract description 3
- 108010001267 Protein Subunits Proteins 0.000 abstract description 3
- 102000002067 Protein Subunits Human genes 0.000 abstract description 2
- 241000709664 Picornaviridae Species 0.000 abstract 2
- 108700028146 Genetic Enhancer Elements Proteins 0.000 abstract 1
- 238000010353 genetic engineering Methods 0.000 abstract 1
- 108020004414 DNA Proteins 0.000 description 38
- 235000018102 proteins Nutrition 0.000 description 37
- 125000003729 nucleotide group Chemical group 0.000 description 28
- 238000001890 transfection Methods 0.000 description 27
- 108090000765 processed proteins & peptides Proteins 0.000 description 21
- 239000002773 nucleotide Substances 0.000 description 20
- 102000004196 processed proteins & peptides Human genes 0.000 description 19
- 239000000499 gel Substances 0.000 description 18
- 238000003752 polymerase chain reaction Methods 0.000 description 18
- 229920001184 polypeptide Polymers 0.000 description 17
- 101710159752 Poly(3-hydroxyalkanoate) polymerase subunit PhaE Proteins 0.000 description 16
- 101710130262 Probable Vpr-like protein Proteins 0.000 description 16
- 210000001519 tissue Anatomy 0.000 description 16
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 15
- 108010020764 Transposases Proteins 0.000 description 14
- 150000001413 amino acids Chemical class 0.000 description 14
- 239000003623 enhancer Substances 0.000 description 14
- 108010021843 fluorescent protein 583 Proteins 0.000 description 14
- 239000000047 product Substances 0.000 description 14
- 101710172711 Structural protein Proteins 0.000 description 13
- 239000012634 fragment Substances 0.000 description 13
- 238000011144 upstream manufacturing Methods 0.000 description 13
- 239000011543 agarose gel Substances 0.000 description 12
- 238000010369 molecular cloning Methods 0.000 description 12
- 108060001084 Luciferase Proteins 0.000 description 11
- 239000005089 Luciferase Substances 0.000 description 11
- 239000002299 complementary DNA Substances 0.000 description 11
- 238000013519 translation Methods 0.000 description 11
- 108091093088 Amplicon Proteins 0.000 description 10
- 241000908128 Plautia stali intestine virus Species 0.000 description 10
- 239000000872 buffer Substances 0.000 description 10
- 238000013518 transcription Methods 0.000 description 10
- 230000035897 transcription Effects 0.000 description 10
- 230000010474 transient expression Effects 0.000 description 10
- 108090000565 Capsid Proteins Proteins 0.000 description 9
- 102000004190 Enzymes Human genes 0.000 description 9
- 108090000790 Enzymes Proteins 0.000 description 9
- 238000010240 RT-PCR analysis Methods 0.000 description 9
- 241000936948 Rhopalosiphum padi virus Species 0.000 description 9
- 102000008579 Transposases Human genes 0.000 description 9
- 238000004113 cell culture Methods 0.000 description 9
- 230000000694 effects Effects 0.000 description 9
- 230000008520 organization Effects 0.000 description 9
- 230000009466 transformation Effects 0.000 description 9
- 101710132601 Capsid protein Proteins 0.000 description 8
- 102100023321 Ceruloplasmin Human genes 0.000 description 8
- 235000001014 amino acid Nutrition 0.000 description 8
- 238000009360 aquaculture Methods 0.000 description 8
- 244000144974 aquaculture Species 0.000 description 8
- 238000000338 in vitro Methods 0.000 description 8
- 108091008146 restriction endonucleases Proteins 0.000 description 8
- 241000907524 Drosophila C virus Species 0.000 description 7
- 101000833492 Homo sapiens Jouberin Proteins 0.000 description 7
- 101000651236 Homo sapiens NCK-interacting protein with SH3 domain Proteins 0.000 description 7
- 102100024407 Jouberin Human genes 0.000 description 7
- 238000010367 cloning Methods 0.000 description 7
- 238000010276 construction Methods 0.000 description 7
- 201000010099 disease Diseases 0.000 description 7
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 7
- 230000002068 genetic effect Effects 0.000 description 7
- 238000001727 in vivo Methods 0.000 description 7
- 239000000203 mixture Substances 0.000 description 7
- 238000000746 purification Methods 0.000 description 7
- 241000238553 Litopenaeus vannamei Species 0.000 description 6
- 101710118046 RNA-directed RNA polymerase Proteins 0.000 description 6
- 230000001580 bacterial effect Effects 0.000 description 6
- 230000001404 mediated effect Effects 0.000 description 6
- 108060004795 Methyltransferase Proteins 0.000 description 5
- 108091034117 Oligonucleotide Proteins 0.000 description 5
- 241000238550 Penaeidae Species 0.000 description 5
- 241000380111 Yellow head virus Species 0.000 description 5
- 230000001413 cellular effect Effects 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 238000011161 development Methods 0.000 description 5
- 230000018109 developmental process Effects 0.000 description 5
- 108020004999 messenger RNA Proteins 0.000 description 5
- 244000052769 pathogen Species 0.000 description 5
- 108091033319 polynucleotide Proteins 0.000 description 5
- 102000040430 polynucleotide Human genes 0.000 description 5
- 239000002157 polynucleotide Substances 0.000 description 5
- 230000002441 reversible effect Effects 0.000 description 5
- 239000000523 sample Substances 0.000 description 5
- 241000894007 species Species 0.000 description 5
- 230000009261 transgenic effect Effects 0.000 description 5
- 241000317943 Acute bee paralysis virus Species 0.000 description 4
- 241000318498 Black queen cell virus Species 0.000 description 4
- 108091026890 Coding region Proteins 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 4
- 241001622355 Himetobi P virus Species 0.000 description 4
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 4
- 241001480150 Triatoma virus Species 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000009313 farming Methods 0.000 description 4
- 235000013305 food Nutrition 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 238000003670 luciferase enzyme activity assay Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 229930182817 methionine Natural products 0.000 description 4
- 210000003205 muscle Anatomy 0.000 description 4
- 230000001052 transient effect Effects 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- 101710094648 Coat protein Proteins 0.000 description 3
- 108020004705 Codon Proteins 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 102100021181 Golgi phosphoprotein 3 Human genes 0.000 description 3
- 108091092195 Intron Proteins 0.000 description 3
- 101710125418 Major capsid protein Proteins 0.000 description 3
- 101710141454 Nucleoprotein Proteins 0.000 description 3
- 241000927735 Penaeus Species 0.000 description 3
- 241000254064 Photinus pyralis Species 0.000 description 3
- 108010076039 Polyproteins Proteins 0.000 description 3
- 101710083689 Probable capsid protein Proteins 0.000 description 3
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 3
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 3
- 108700008625 Reporter Genes Proteins 0.000 description 3
- 108091081024 Start codon Proteins 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 230000007613 environmental effect Effects 0.000 description 3
- 230000002538 fungal effect Effects 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 230000036541 health Effects 0.000 description 3
- 230000001939 inductive effect Effects 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 230000000977 initiatory effect Effects 0.000 description 3
- 238000003780 insertion Methods 0.000 description 3
- 230000037431 insertion Effects 0.000 description 3
- 230000010354 integration Effects 0.000 description 3
- 239000006166 lysate Substances 0.000 description 3
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 3
- 230000001766 physiological effect Effects 0.000 description 3
- -1 rRNA Proteins 0.000 description 3
- 230000022532 regulation of transcription, DNA-dependent Effects 0.000 description 3
- 238000010561 standard procedure Methods 0.000 description 3
- 238000011282 treatment Methods 0.000 description 3
- 208000035143 Bacterial infection Diseases 0.000 description 2
- 101100297347 Caenorhabditis elegans pgl-3 gene Proteins 0.000 description 2
- 102000016911 Deoxyribonucleases Human genes 0.000 description 2
- 108010053770 Deoxyribonucleases Proteins 0.000 description 2
- 208000035240 Disease Resistance Diseases 0.000 description 2
- 241000255352 Drosophila virilis Species 0.000 description 2
- 102100031780 Endonuclease Human genes 0.000 description 2
- 108091060211 Expressed sequence tag Proteins 0.000 description 2
- 241001235208 Farfantepenaeus paulensis Species 0.000 description 2
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 2
- 241000530454 Litopenaeus schmitti Species 0.000 description 2
- 208000031888 Mycoses Diseases 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 108091005461 Nucleic proteins Proteins 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 108091093037 Peptide nucleic acid Proteins 0.000 description 2
- 241001527104 Plautia stali Species 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- 108091081062 Repeated sequence (DNA) Proteins 0.000 description 2
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 108010017842 Telomerase Proteins 0.000 description 2
- 108091036066 Three prime untranslated region Proteins 0.000 description 2
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 2
- 235000004279 alanine Nutrition 0.000 description 2
- 230000000692 anti-sense effect Effects 0.000 description 2
- 230000000840 anti-viral effect Effects 0.000 description 2
- 239000004599 antimicrobial Substances 0.000 description 2
- 210000000234 capsid Anatomy 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 230000009089 cytolysis Effects 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
- 230000037430 deletion Effects 0.000 description 2
- 238000001976 enzyme digestion Methods 0.000 description 2
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 2
- 229960005542 ethidium bromide Drugs 0.000 description 2
- 230000007717 exclusion Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000012252 genetic analysis Methods 0.000 description 2
- 210000003677 hemocyte Anatomy 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 230000000366 juvenile effect Effects 0.000 description 2
- 238000012423 maintenance Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 235000019419 proteases Nutrition 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 210000003705 ribosome Anatomy 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 239000013535 sea water Substances 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 230000009447 viral pathogenesis Effects 0.000 description 2
- 210000002845 virion Anatomy 0.000 description 2
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- CNNSWSHYGANWBM-UHFFFAOYSA-N 6-chloro-2,3-dimethylquinoxaline Chemical compound C1=C(Cl)C=C2N=C(C)C(C)=NC2=C1 CNNSWSHYGANWBM-UHFFFAOYSA-N 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 102000007469 Actins Human genes 0.000 description 1
- 241000702419 Ambidensovirus Species 0.000 description 1
- 241000256837 Apidae Species 0.000 description 1
- 241000238421 Arthropoda Species 0.000 description 1
- 241000238017 Astacoidea Species 0.000 description 1
- 101000666833 Autographa californica nuclear polyhedrosis virus Uncharacterized 20.8 kDa protein in FGF-VUBI intergenic region Proteins 0.000 description 1
- 101000977023 Azospirillum brasilense Uncharacterized 17.8 kDa protein in nodG 5'region Proteins 0.000 description 1
- 101000977027 Azospirillum brasilense Uncharacterized protein in nodG 5'region Proteins 0.000 description 1
- 101000962005 Bacillus thuringiensis Uncharacterized 23.6 kDa protein Proteins 0.000 description 1
- 101000961984 Bacillus thuringiensis Uncharacterized 30.3 kDa protein Proteins 0.000 description 1
- 241000965621 Brevidensovirus Species 0.000 description 1
- 102100025933 Cancer-associated gene 1 protein Human genes 0.000 description 1
- 241001289493 Cripavirus Species 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
- 101000644901 Drosophila melanogaster Putative 115 kDa protein in type-1 retrotransposable element R1DM Proteins 0.000 description 1
- 101000785191 Drosophila melanogaster Uncharacterized 50 kDa protein in type I retrotransposable element R1DM Proteins 0.000 description 1
- 241000255348 Drosophila sp. (in: Insecta) Species 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 101000747704 Enterobacteria phage N4 Uncharacterized protein Gp1 Proteins 0.000 description 1
- 101000747702 Enterobacteria phage N4 Uncharacterized protein Gp2 Proteins 0.000 description 1
- 101000861206 Enterococcus faecalis (strain ATCC 700802 / V583) Uncharacterized protein EF_A0048 Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 101000769180 Escherichia coli Uncharacterized 11.1 kDa protein Proteins 0.000 description 1
- 101000758599 Escherichia coli Uncharacterized 14.7 kDa protein Proteins 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 108091029865 Exogenous DNA Proteins 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- 241001235206 Farfantepenaeus brasiliensis Species 0.000 description 1
- 241000694574 Farfantepenaeus californiensis Species 0.000 description 1
- 241000927582 Farfantepenaeus subtilis Species 0.000 description 1
- 241000995704 Fenneropenaeus chinensis Species 0.000 description 1
- 241001149925 Fenneropenaeus indicus Species 0.000 description 1
- 241001235204 Fenneropenaeus merguiensis Species 0.000 description 1
- 241000335026 Fenneropenaeus penicillatus Species 0.000 description 1
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 1
- 101000933825 Homo sapiens Cancer-associated gene 1 protein Proteins 0.000 description 1
- 101000768930 Lactococcus lactis subsp. cremoris Uncharacterized protein in pepC 5'region Proteins 0.000 description 1
- 241000254158 Lampyridae Species 0.000 description 1
- 101000976301 Leptospira interrogans Uncharacterized 35 kDa protein in sph 3'region Proteins 0.000 description 1
- 101000976302 Leptospira interrogans Uncharacterized protein in sph 3'region Proteins 0.000 description 1
- 101000778886 Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai (strain 56601) Uncharacterized protein LA_2151 Proteins 0.000 description 1
- 241000530452 Litopenaeus Species 0.000 description 1
- 241000870576 Litopenaeus occidentalis Species 0.000 description 1
- 241000907643 Litopenaeus setiferus Species 0.000 description 1
- 241001157775 Litopenaeus stylirostris Species 0.000 description 1
- 241001124325 Marsupenaeus japonicus Species 0.000 description 1
- 241000335017 Melicertus latisulcatus Species 0.000 description 1
- 241001454399 Metapenaeus Species 0.000 description 1
- 241000331928 Metapenaeus affinis Species 0.000 description 1
- 241001600647 Metapenaeus dobsoni Species 0.000 description 1
- 241001286665 Metapenaeus monoceros Species 0.000 description 1
- 108091092878 Microsatellite Proteins 0.000 description 1
- 101000658690 Neisseria meningitidis serogroup B Transposase for insertion sequence element IS1106 Proteins 0.000 description 1
- 241001484257 Nimaviridae Species 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 108010038807 Oligopeptides Proteins 0.000 description 1
- 102000015636 Oligopeptides Human genes 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 108091081548 Palindromic sequence Proteins 0.000 description 1
- 241000726026 Parsnip yellow fleck virus Species 0.000 description 1
- 241000701945 Parvoviridae Species 0.000 description 1
- 241000238552 Penaeus monodon Species 0.000 description 1
- 241000907637 Penaeus semisulcatus Species 0.000 description 1
- 241000238554 Penaeus sp. Species 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- 108020005089 Plant RNA Proteins 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- 101000748660 Pseudomonas savastanoi Uncharacterized 21 kDa protein in iaaL 5'region Proteins 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 108010081734 Ribonucleoproteins Proteins 0.000 description 1
- 102000004389 Ribonucleoproteins Human genes 0.000 description 1
- 108010041388 Ribonucleotide Reductases Proteins 0.000 description 1
- 102000000505 Ribonucleotide Reductases Human genes 0.000 description 1
- 101000584469 Rice tungro bacilliform virus (isolate Philippines) Protein P1 Proteins 0.000 description 1
- 101001121571 Rice tungro bacilliform virus (isolate Philippines) Protein P2 Proteins 0.000 description 1
- 241000293871 Salmonella enterica subsp. enterica serovar Typhi Species 0.000 description 1
- 108091061939 Selfish DNA Proteins 0.000 description 1
- 238000002105 Southern blotting Methods 0.000 description 1
- 101000818096 Spirochaeta aurantia Uncharacterized 15.5 kDa protein in trpE 3'region Proteins 0.000 description 1
- 101000818098 Spirochaeta aurantia Uncharacterized protein in trpE 3'region Proteins 0.000 description 1
- 101000766081 Streptomyces ambofaciens Uncharacterized HTH-type transcriptional regulator in unstable DNA locus Proteins 0.000 description 1
- 101001026590 Streptomyces cinnamonensis Putative polyketide beta-ketoacyl synthase 2 Proteins 0.000 description 1
- 108091027544 Subgenomic mRNA Proteins 0.000 description 1
- 101000804403 Synechococcus elongatus (strain PCC 7942 / FACHB-805) Uncharacterized HIT-like protein Synpcc7942_1390 Proteins 0.000 description 1
- 101000750910 Synechococcus elongatus (strain PCC 7942 / FACHB-805) Uncharacterized HTH-type transcriptional regulator Synpcc7942_2319 Proteins 0.000 description 1
- 101000750896 Synechococcus elongatus (strain PCC 7942 / FACHB-805) Uncharacterized protein Synpcc7942_2318 Proteins 0.000 description 1
- 101000644897 Synechococcus sp. (strain ATCC 27264 / PCC 7002 / PR-6) Uncharacterized protein SYNPCC7002_B0001 Proteins 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 108700019146 Transgenes Proteins 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 108091023045 Untranslated Region Proteins 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 241001058063 Whispovirus Species 0.000 description 1
- 101000916321 Xenopus laevis Transposon TX1 uncharacterized 149 kDa protein Proteins 0.000 description 1
- 101000916336 Xenopus laevis Transposon TX1 uncharacterized 82 kDa protein Proteins 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 101001000760 Zea mays Putative Pol polyprotein from transposon element Bs1 Proteins 0.000 description 1
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 101000760088 Zymomonas mobilis subsp. mobilis (strain ATCC 10988 / DSM 424 / LMG 404 / NCIMB 8938 / NRRL B-806 / ZM1) 20.9 kDa protein Proteins 0.000 description 1
- 101000678262 Zymomonas mobilis subsp. mobilis (strain ATCC 10988 / DSM 424 / LMG 404 / NCIMB 8938 / NRRL B-806 / ZM1) 65 kDa protein Proteins 0.000 description 1
- 230000001594 aberrant effect Effects 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000003466 anti-cipated effect Effects 0.000 description 1
- PXXJHWLDUBFPOL-UHFFFAOYSA-N benzamidine Chemical compound NC(=N)C1=CC=CC=C1 PXXJHWLDUBFPOL-UHFFFAOYSA-N 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000004590 computer program Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000005860 defense response to virus Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000006806 disease prevention Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000009509 drug development Methods 0.000 description 1
- 241001493065 dsRNA viruses Species 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 108091006047 fluorescent proteins Proteins 0.000 description 1
- 102000034287 fluorescent proteins Human genes 0.000 description 1
- 210000004602 germ cell Anatomy 0.000 description 1
- 229940000351 hemocyte Drugs 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 241000238565 lobster Species 0.000 description 1
- 235000009973 maize Nutrition 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 210000004779 membrane envelope Anatomy 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000011278 mitosis Effects 0.000 description 1
- 239000003607 modifier Substances 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000000816 peptidomimetic Substances 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 238000013081 phylogenetic analysis Methods 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 230000001323 posttranslational effect Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000004224 protection Effects 0.000 description 1
- 108060006633 protein kinase Proteins 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 230000003362 replicative effect Effects 0.000 description 1
- 210000001995 reticulocyte Anatomy 0.000 description 1
- 230000001177 retroviral effect Effects 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 229940098714 salmonella enterica subsp. enterica serovar typhi Drugs 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 235000002639 sodium chloride Nutrition 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- PIEPQKCYPFFYMG-UHFFFAOYSA-N tris acetate Chemical compound CC(O)=O.OCC(N)(CO)CO PIEPQKCYPFFYMG-UHFFFAOYSA-N 0.000 description 1
- 241000701447 unidentified baculovirus Species 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 244000052613 viral pathogen Species 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
- A01K67/0333—Genetically modified invertebrates, e.g. transgenic, polyploid
- A01K67/0337—Genetically modified Arthropods
- A01K67/0338—Genetically modified Crustaceans
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/70—Invertebrates
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/02—Animal zootechnically ameliorated
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/0393—Animal model comprising a reporter system for screening tests
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/90—Vectors containing a transposable element
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2830/00—Vector systems having a special element relevant for transcription
- C12N2830/60—Vector systems having a special element relevant for transcription from viruses
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2840/00—Vectors comprising a special translation-regulating system
- C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
- C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES
Definitions
- WSSV white spot syndrome virus
- YHV yellowhead virus
- TSV Taura syndrome virus
- IHHNV infectious hypodermal and hematopoietic necrosis virus
- vectors could be used (1) to express recombinant protein(s) with therapeutic potential using shrimp or other crustacean host, (2) to express host gene or foreign gene in excess to determine their role in growth, development, and or disease resistance using shrimp or other crustacean host, (3) to develop a transgenic shrimp or other crustaceans, and (4) to study the role of virally encoded protein in viral pathogenesis in vitro and in vivo.
- These techniques will develop the tools needed to modify primary cell cultures to enable the development of unregulated growth necessary for immortalized cell lines.
- the inventors have discovered a number of important tools that will enable the manipulation of animal genes both transiently and permanently in both tissue culture and live animals.
- the invention harnesses elements from shrimp viruses that through bioinformatics evaluation have been shown to have homology to enhancer or regulatory elements in other species.
- the inventors have demonstrated specific vectors that can be developed from these elements and methods of their use in the instant invention that will allow the improvement of animal health as well as diagnostic uses thereof.
- the invention relates to improvement of crustacean and especially shrimp health in relation to virally transmitted disease.
- the invention also provides tools for drug development and diagnostics of the same.
- transfection vector for developing transgenic fish, shellfish or insects expressing a foreign gene or genes. Such a vector would have a broad host range that may be applicable to other animals as an expression system.
- pathogen-encoded protein e.g., capsid protein
- pathogen- encoded protein or proteins e.g., capsid protein, enzyme, receptor, recognition sequence
- Figure 1 consists of Figures 1A, IB, IC, and ID. A comparative representation of the genomic organization of several mammalian and insect picomaviruses and plant RNA viruses - PV in Figure 1A, IFV in Figure IB, TSV in Figure IC, and PYFV in Figure ID.
- ORF Open reading frame
- UTR untranslated region
- VPg genome linked protein
- ? the presence of VPg has not been confirmed.
- the helicase (triangle), protease (circle), and the RNA dependent RNA polymerase (square) regions are indicated.
- Figure 2 consists of Figures 2A, 2B, and 2C.
- the intergenic regions between ORF1 and ORF 2 of TSV are aligned and compared with the homologous regions of insect picorna-like viruses.
- the sequences in Figure 2A continue in Figure 2B, and those in Figure 2B continue in Figure 2C.
- Nucleotide residues indicated in bold, underlined text are conserved residues, underlined (but not bold) residues are partially conserved among the sequences, and residues indicated in ordinary text appear not to be conserved.
- SEQ ID NOs: are, for cricket paralysis viruse (CrPV), SEQ ID NO: 1; for Drosophila C virus (DCV), SEQ ID NO: 2; for Plautia stali intestine virus (PSIV), SEQ ID NO: 3; for Himetobi P virus (HiPV), SEQ ID NO: 4; for black queen cell virus (BQCV), SEQ ID NO: 5; for triatoma virus (TrV), SEQ ID NO: 6; for Rhopalosiphum padi virus (RhPV), SEQ ID NO: 7; for Taura syndrome virus (TSV), SEQ ID NO: 8; for acute bee paralysis virus (ABPV), SEQ ID NO: 9.
- Figure 3 is a schematic representation of the genome organization of IHHNV, based on the sequence of GenBank accession no. AF273215.
- the numbers on the diagram indicate nucleotide residue numbers.
- the left open reading frame (ORF) starts at nucleotide residue 313 and ends at residue 2596
- the middle ORF starts at nucleotide residue 534 and ends at residue 1631
- the right ORF starts at nucleotide residue 2535 and ends at residue 3527.
- the IHHNV promoters, P2 and P61 are indicated by open triangles.
- Figure 4 is an image of results of an agarose gel electrophoreses of the IHHNV P61 amplicon.
- Figure 5 is a circular nucleotide map of the WSSV D ⁇ A genome (Thai-isolate, GenBank accession no. AF369029). The nucleotide residue designated 1 is the nucleotide A of ATG codon of the capsid protein gene VP28. The arrangement of all putative genes in both strands is shown by boxes on the ring inside the scale.
- Bars on the dashed line indicate the location of the repeat regions throughout the genome.
- the locations of the viral capsid genes (VP) are indicated by bars on the dotted line in the map.
- the ring within the ring indicating viral capsid genes indicates the GC skew, and the ring in the center represents G skew.
- Figure 6 is a BLASTX search and amino acid alignment of WSSV transposase amino acid sequence (SEQ ID NO: 10; WSSVT is the transposase of WSSV-Taiwan isolate ORF 166) and a bacterial transposon amino acid sequence (SEQ ID NO: 11; SESET is a transposon of Salmonella enterica subsp. enterica serovar Typhi; GenBank Accession no. 16760800).
- FIG. 7 is a schematic map of the transposase gene in WSSV isolate Taiwan (GenBank accession number AF440570). Inverted repeat sequences are indicated by the abbreviation IR. Viral repeat sequence (SEQ ID NO: 12) is indicated in the Repeat box. The numbers on top of the boxes (238587 and 239925) indicate the nucleotide position in the genome of the WSSV-Taiwan isolate. The length 1337 bp indicates the length of transposase gene, in base pairs. The palindromic sequence within the inverted repeat sequences (SEQ ID NOs: 13 and 14) is underlined.
- Figure 8 is a BLASTX search and amino acid sequence alignment of shrimp DNA clone AF077579 amino acid sequence (SEQ ID NO: 15; "Shrimp”; Xu et al., 1999, Animal Genet. 30:1-7) and the non-LTR Penelope transposable element of Drosophila virilis amino acid sequence (SEQ ID NO: 16; from GenBank Accession no. Q24736).
- Figure 9A and 9B is a schematic representation of shrimp expression vectors described herein.
- the vector diagramed in Figure 9A is a transient expression vector containing the IHHNV P61 promoter, IRES element of the TSV intergenic region between ORF1 and ORF2, and a marker gene.
- the vector diagramed in Figure 9B is a transfection vector for shrimp containing the IHHNV P61 promoter, shrimp Penelope full-length ORF, TSV IRES element and a selectable marker (e.g., pDsRed).
- Shrimp Penelope represents the full-length copy of the Penelope gene from shrimp. Arrows marked by 5' and 3' represent the positions of the shrimp Penelope element inverted terminal repeats.
- FIG 10 is a schematic representation of a shrimp transfection expression vector described herein.
- a transfection vector for shrimp can contain the IHHNV P61 promoter, full-length WSSV transposase gene, TSV IRES element and a selectable marker
- Arrows marked by 5 1 and 3' represent the positions of the WSSV inverted terminal repeats.
- Figure 11 is a map of a plasmid constructed by inserting the IHHNV p61 promoter into the pGL3-Basic vector (Promega).
- Figure 12 is an image of an agarose gel separation of plasmids produced in
- Lane 1 contains a 1 kilobase DNA standard
- Lanes 2-5 contain linearized pGL3 enhancer vector
- Lane 6 contains a 100 base pair DNA standard
- Lanes 8-11 contain individual pSTF isolates double digested with S cl and Nbel.
- Figure 13 is a map of a pSTH vector constructed using the IHH ⁇ V p61 promoter and the pGL3- Enhancer vector as a backbone.
- Figure 14 is a map of a pSTI vector constructed using the IHH ⁇ V p2 promoter and the pGL3- Enhancer vector as a backbone.
- Figure 15 consisting of Figures 15A and 15B, is a pair of plasmid maps of
- IHH ⁇ V p61 promoter driving DsRed2 expression without added IRES element pSTD, shown in Figure 15 A
- TSV IRES element pSTE, shown in Figure 15B
- Figure 16 is an image of an agarose gel separation of the digest described in
- Lane 1 contains a 100 base pair D ⁇ A ladder standard.
- Lane 2-TSV contains the IRES PCR product.
- Figure 17 is a standard curve generated for the luciferase assay used to determine expression of luciferase in shrimp tissues, as described in Example 17 herein.
- the invention relates to compositions and methods that can be used to express heterologous nucleic acids and proteins in cells of marine creatures, including crustaceans such as shrimp, lobster, crayfish, and crabs, and further including non-crustacean arthropods and other marine animals such as fish and mollusks.
- the invention relates to a non-naturally-occurring nucleic acid construct that includes at least a viral promoter region and an insect picomavirus (IPV) internal ribosome entry site (IRES).
- IPV insect picomavirus
- IRES internal ribosome entry site
- One or both of the promoter and the IRES can be linked with one or more cistrons, such as an intron-free open reading frame (ORF).
- ORF intron-free open reading frame
- virus promoter and an IRES from an IPV renders the construct capable of expressing in a crustacean cell RNA or protein encoded by a nucleic acid introduced into the cell as part of the construct.
- Such constructs can be used to produce one or more nucleic acids in a host crustacean cell.
- an mRNA encoding a protein of interest can be produced, as can an RNA that binds with a specific endogenously- produced nucleic acid of the cell.
- a protein encoded by a portion of the construct can produced in a crustacean cell by incorporating into the cell a construct in which the portion is operably linked to a viral promoter to generate RNA that includes both the portion and an IPV IRES, which can be translated by the host cell to produce the protein.
- the identities of the virus from which the promoter and the IPV from which the IRES are derived is not critical.
- the promoter and the IRES are derived from the same IPV, but elements from distinct viruses can be used as well.
- the IPV(s) can be substantially any IPV that is currently known or hereafter developed.
- a preferred type of IPV is the class of IPVs that are designated "cricket paralysis-like" picomaviruses, as described in the art (e.g., Christian et al., 1998, In: Miller et al., Eds., "The Insect Viruses" New York, Plenum Press, pp.
- IPV includes both viruses that are capable of infecting hosts in phylum Arthropoda, regardless of whether they are designated "picomaviruses" or "picorna-like viruses.”
- Knowledge of the identity of a host cell into which a nucleic acid construct of the type described herein will be incorporated can guide selection of an appropriate IPV. It has been discovered that promoters from substantially any virus (at least viruses suitable for any actual or anticipated host for the construct) and IRES' from substantially any IPV can be used in vectors intended for use in crustaceans.
- a promoter, an IRES, or both from a virus that is known to infect crustaceans of the same species as the crustacean from which the host cell was derived.
- a promoter and IRES from among those of known shrimp viruses, such as infectious hypodermal and hematopoietic necrosis virus (IHHNV), Taura syndrome virus (TSV), or white spot syndrome virus (WSSV).
- IHHNV infectious hypodermal and hematopoietic necrosis virus
- TSV Taura syndrome virus
- WSSV white spot syndrome virus
- any viral promoter can be used in the nucleic acid constructs described herein.
- suitable promoters include promoters from TSV, IHHNV, and WSSV (e.g., the P2 or P61 promoter of WSSV).
- an IRES occurs in the 5'-untranslated region of the viral genome.
- an IRES occurs in the intergenic region between two or more cistronic regions of the viral genome. Either type of IRES can be incorporated into the nucleic acid construct described herein.
- the nucleic acid constructs described herein can be designed to express a product for a relatively short time or for a longer period, such as the lifetime of a host cell or organism, or even across generations of organisms.
- the association between the form of a nucleic acid construct (e.g., straight-chain nucleic acid, plasmid, or viral vector) and its duration in vivo are known and a skilled artisan can influence the duration of product production from the construct by selection of a suitable form of the construct.
- Duration of product production can also be influenced by the properties of the genetic elements included in the construct.
- the promoter that is used can be an inducible or repressible promoter, subject to regulation by its effectors.
- promoters can induce transcription of an operably linked sequence at a high or low level, depending on the identity of the promoter and the host organism.
- Residence in a host cell of a construct described herein can be influenced by the presence or absence of genetic elements in the construct.
- the construct in introduced into a host cell in the form of a plasmid the plasmid (together with the construct) will be replicated if the plasmid includes an origin of replication site (ORS) that is operable in the host cell.
- ORS origin of replication site
- ORS can induce maintenance of single, a few, or multiple copies of a plasmid in a cell, and a skilled artisan can select an appropriate ORS for a desired copy number for a desired host cell. Further by way of example, if the construct is included in a virus vector construct that is capable of replication and packaging (or can be induced to replicate and package), then maintenance or spread of the construct can be controlled using methods understood by a skilled artisan in this field.
- One or more cistronic elements e.g., an ORF
- IRES an ORF
- the cistronic element should also be operably linked with the promoter, so as to enhance transcription of RNA encoding the cistron prior to translation brought about by introduction of a ribosome into the RNA by way of the IRES (or by a normal, mRNA cap-targeted ribosome or both).
- the cistron is preferably an ORF that is not interrupted by an intron, but one or more introns can be present so long as the host cell is able to remove the intron(s) to yield the construct-encoded product of interest.
- a cistron of the construct can encode an RNA that has a physiological effect on the host cell (e.g., one that hybridizes specifically with a nucleic acid in the cell).
- the cistron can also encode a protein.
- the cell is a cell of a crustacean food product (e.g., a farm-raised shrimp)
- the protein can be one which enhances the human nutritional value, the health of the crustacean, or the disease resistance of the crustacean. Proteins which enhance the lifespan, physiology or rate of proliferation of an individual cell are further examples of proteins that can be expressed in a host cell from the nucleic acid construct described herein.
- a protein or nucleic acid expressed in a cell can exert a direct physiological effect on a cell (e.g., a protein that catalyzes a chemical reaction or is a structural component of a cell or a nucleic acid that binds with a cellular nucleic acid in way that affects expression of the cellular nucleic acid). It is also known that a protein or nucleic acid expressed in a cell can exert a relatively indirect physiological effect on a cell (e.g., a protein that acts as a transcription factor or a nucleic acid that modulates the activity of a cellular nucleic acid polymerase).
- nucleic acid construct described herein when the construct is introduced into a host cell.
- a skilled artisan can select an appropriate product based on the identity and biochemical properties of the host cell and the desired effect, and no more than ordinary experimentation is required to make a construct that produces the appropriate product.
- Such products can be products that are ordinarily produced in the host cell (e.g., at a different level in the presence and absence of the construct) or can be heterologous products (e.g., products unlike any produced by the host cell or an altered form of a homologous product).
- a transposable element can be included in the construct if desired.
- a TE facilitates distribution of operably linked elements of the construct into the genome of the host cell.
- a TE can thereby incorporate a transgene from the construct described herein (transiently or relatively permanently, depending on the TE selected) into the host genome.
- a skilled artisan is able to operably link selected elements of the construct described herein with a TE with no more than routine experimentation.
- suitable TEs include those that are homologous to the Drosophila Penelope TE sequence reported in the literature.
- Other suitable TEs include IPV TEs, such as those of WSSV.
- the nucleic acid construct described herein can be used to express a protein in a host cell, such as a crustacean cell. This is achieved by introducing into the host cell one or more copies of the nucleic acid construct described herein.
- the method by which the construct is introduced into the host cell is not critical, and a variety of methods of introducing a nucleic acid vector into a cell are known in the art. Examples of suitable methods of incorporating the construct into a host cell include use of a virus vector containing the construct, a ballistically administered particle coated with or containing the construct, and electroporesis of cells in the presence of a plasmid that includes the construct.
- “Shrimp” refers to any of the group of crustaceans that are commonly cultured for aquaculture or captured in the wild fisheries.
- the term “shrimp” includes shrimp eggs, shrimp larvae, shrimp post-larvae and adult shrimp.
- the term “shrimp” and “prawn” will be used interchangeably throughout the specification.
- Shrimp can refer to but are not limited to, Penaeus shrimp and include the species Penaeus vannamei, Penaeus chinensis, Penaeus monodon, Penaeus stylirostris, Penaeus japonicus, Penaeus penicillatus, Penaeus merguiensis, Penaeus indicus, Penaeus subtilis, Penaeus paulensis, Penaeus setiferus, Penaeus brasiliensis, Penaeus duorarum, Penaeus occidentalis, Penaeus schmitti, Penaeus calif orniensis, Penaeus semisulcatus, Penaeus latisulcatus, Metapenaeus monoceros, Metapenaeus dobsoni, Metapenaeus affinis, and Metapenaeus brivicornis; and Litopenaeid shrimp (such as Litopenaeus vannamei, L. setiferus).
- a "vector” comprises a nucleic acid, which can infect, transfect, and transiently or permanently transform or transfect a cell. It will be recognized that a vector can be a naked nucleic acid, or a nucleic acid complexed with protein or lipid.
- the vector optionally comprises viral or bacterial nucleic acids and/or proteins, and/or membranes (e.g., a cell membrane, a viral lipid envelope, etc.).
- Vectors include, but are not limited to replicons (e.g., RNA replicons, bacteriophages) to which fragments of DNA may be attached and become replicated.
- Vectors thus include, but are not limited to RNA, autonomous self- replicating circular or linear DNA or RNA (e.g., plasmids, viruses, and the like, see, e.g., U.S. Patent No. 5,217,879), and include both the expression and non-expression plasmids.
- RNA autonomous self- replicating circular or linear DNA or RNA
- plasmids plasmids, viruses, and the like, see, e.g., U.S. Patent No. 5,217,879
- a recombinant microorganism or cell culture is described as hosting an "expression vector” this includes both extra-chromosomal circular and linear DNA and DNA that has been incorporated into the host chromosome(s).
- the vector may either be stably replicated by the cells during mitosis as an autonomous structure, or is incorporated within the host's genome.
- promoter includes all sequences capable of driving transcription of a coding sequence in a cell, e.g., a plant cell, animal cell, bacterial cell, fungal cell, and yeast cell.
- promoters used in the constructs of the invention include cis-acting transcriptional control elements and regulatory sequences that are involved in regulating or modulating the timing and/or rate of transcription of a gene or genes.
- a promoter can be a cw-acting transcriptional control element, including an enhancer, a promoter, a transcription terminator, an origin of replication, a chromosomal integration sequence, 5' and 3' untranslated regions, or an intronic sequence, which are involved in transcriptional regulation.
- cis-ac ting sequences typically interact with proteins or other biomolecules to carry out (turn on/ off, regulate, modulate, etc.) transcription.
- Constutive promoters are those that drive expression continuously under most environmental conditions and states of development or cell differentiation.
- Inducible or “regulatable” promoters direct expression of the nucleic acid of the invention under the influence of environmental conditions or developmental conditions. Examples of environmental conditions that may affect transcription by inducible promoters include anaerobic conditions, elevated temperature, drought, or the presence of light.
- “Plasmids” can be commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures. Equivalent plasmids to those described herein are known in the art and will be apparent to the ordinarily skilled artisan.
- Transposable elements are a group of genetic elements that move from one locus to another by non-homologous recombination.
- Internal ribosome entry site or “IRES” refers to a translation control element for cap independent mRNA translation. IRES' have been described in the art (e.g., see review by Hellen et al., 2001, Genes Develop. 15:1596-1612), and a skilled artisan is able to operably link an open reading frame or cistron with an IRES with no more than routine experimentation.
- the term "gene” includes a nucleic acid sequence comprising a segment of DNA involved in producing a transcription product (e.g., a message), which in turn is translated to produce a polypep tide chain, or regulates gene transcription, reproduction or stability. Genes can include regions preceding and following the coding region, such as leader and trailer, promoters and enhancers, as well as, where applicable, intervening sequences (introns) between individual coding segments (exons).
- nucleic acid or “nucleic acid sequence” includes oligonucleotide, nucleotide, polynucleotide, or to a fragment of any of these, to DNA or RNA (e.g., mRNA, rRNA, tRNA) of genomic or synthetic origin which may be single-stranded (ss) or double- stranded (ds) and may represent a sense (+) or antisense (-) strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material, natural or synthetic in origin, including, e.g., iRNA, ribonucleoproteins (e.g., iRNPs).
- DNA or RNA e.g., mRNA, rRNA, tRNA
- PNA peptide nucleic acid
- DNA-like or RNA-like material natural or synthetic in origin, including, e.g., iRNA, ribonucleoproteins (e.g
- nucleic acids i.e., oligonucleotides, containing known analogues of natural nucleotides.
- the term also encompasses nucleic-acid-like structures with synthetic backbones (see e.g., Mata, 1997, Toxicol. Appl. Pharmacol. 144:189-197; Strauss-Soukup, 1997, Biochemistry 36:8692- 8698; Strauss-Soukup, 1997, Biochemistry 36:8692- 8698; Mull, 1996, Antisense Nucl. Acid Drug Dev. 6:153-156).
- amino acid or “amino acid sequence” includes an oligopeptide, peptide, polypeptide, or protein sequence, or to a fragment, portion, or subunit of any of these, and to naturally occurring or synthetic molecules.
- polypeptide and “protein” include amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain modified amino acids other than the 20 gene-encoded amino acids.
- polypeptide also includes peptides and polypeptide fragments, motifs and the like. The term also includes glycosylated polypeptides.
- the peptides and polypep tides of the invention also include all “mimetic” and “peptidomimetic” forms, as described in further detail, below.
- isolated includes a material removed from its original environment, e.g., the natural environment if it is naturally occurring.
- a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated.
- Such polynucleo tides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.
- an isolated material or composition can also be a "purified" composition, i.e., it does not require absolute purity; rather, it is intended as a relative definition.
- Individual nucleic acids obtained from a library can be conventionally purified to electrophoretic homogeneity.
- the invention provides nucleic acids, which have been purified, from genomic DNA or from other sequences in a library or other environment by at least one, two, three, four, five, or more orders of magnitude.
- nucleic acids can include nucleic acids adjacent to a "backbone” nucleic acid to which it is not adjacent in its natural environment.
- nucleic acids represent 5% or more of the number of nucleic acid inserts in a population of nucleic acid "backbone molecules.”
- Backbone molecules include nucleic acids such as expression vectors, self-replicating nucleic acids, viruses, integrating nucleic acids, and other vectors or nucleic acids used to maintain or manipulate a nucleic acid insert of interest.
- the (isolated, recombinant, enriched) nucleic acids represent 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more of the number of nucleic acid inserts in the population of recombinant backbone molecules.
- Recombinant polypeptides or proteins refer to polypeptides or proteins produced by recombinant DNA techniques; e.g., produced from cells transformed by an exogenous DNA construct encoding the desired polypeptide or protein.
- synthetic polypeptides or protein are those prepared by chemical synthesis, as described in further detail, below.
- a promoter sequence can be "operably linked to" a coding sequence when RNA polymerase which initiates transcription at the promoter will transcribe the coding sequence into mRNA, as discussed further, below.
- Primer includes either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands, which may be chemically synthesized. Such synthetic oligonucleotides have no 5' phosphate and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide can ligate to a fragment that has not been dephosphorylated. [0065] All other terms are defined in the literature using Sambrook (Sambrook et al, 1989, "Molecular Cloning: A laboratory manual” Ed. 2, Cold Spring Harbor Press, Cold Spring Harbor) as a guide.
- the TSV genome is a single-stranded RNA of positive polarity with a 3'-poly(A) tail (Bonami et al, 1997, J. Gen. Virol. 78:313-319).
- the genome is 10205 nucleotides (nt) long with a 5' untranslated region of 377 nt and a 3' untranslated region of 226 nt (Mari et al. 2002).
- ORFs open reading frames in the TSV genome.
- ORF1 is 6324 nt long, and encodes a 2107 amino acid (aa) polyprotein with a molecular mass of 234 kDa.
- ORF2 is 3036 nt long and encodes a 1011 aa polypeptide with a molecular mass of 112 kDa (Mari et al., 2002, J. Gen. Virol. 83:915-926; Robles-Sikisaka et al., 2001, Arch. Virol.l46:941-952). There is an intergenic region of 226 nt between the two ORFs.
- ORF1 encodes non-structural proteins (helicase, a protease and a RNA-dependent RNA polymerase, RdRp), and ORF2 encodes the virion structural proteins (Fig. 1).
- TSV virions contain three major polypeptides, designated as VP1, VP2, and VP3 (55, 40, and 24 kDa), and one minor polypeptide (58 kDa), designated as VPO (Bonami et al., 1997, J. Gen. Virol. 78:313-319).
- the N-termini of VP1 to VP3 have been sequenced, and the order of these proteins in ORF2 was found to be VP2, VP1 and VP3 (Mari et al., 2002, J. Gen. Virol. 83:915-926).
- VPO The N-terminal sequence of VPO has not been determined, but it has been hypothesized that it might be processed from ORF2 in a manner similar to PSIV, an insect picorna-like virus infecting the brown-winged green bug, Plautia stali (Sasaki et al., 1998, Virology 244:50-58).
- the VP2/VP1 cleavage site in TSV is conserved in insect picomaviruses: TSV (GF ⁇ SKD), Plautia stali intestine virus, PSIV (GF ⁇ SKP), Drosophila C virus, DCV (GF ⁇ SKP) and Rhophalosiphum padi virus, RhPV (GW ⁇ SKP) (Robles- Sikisaka et al., 2001, Arch. Virol.l46:941-952).
- the presumed VP1 and VP3 cleavage site in TSV (H . ⁇ ) is partially conserved with those used by insect picomaviruses Q ⁇ (A,S,V) (Robles-Sikisaka et al., 2001, Arch. Virol.l46:941-952).
- Picomaviruses have been isolated from a wide range of insect species. Based on their biological, biophysical properties, and genome organization data these viruses were classified as a member of a newly designated group, "Cricket paralysis-like viruses” in Picomaviridae with CrPV as the type species of this group (Christian et al., 1998, In: Miller et al., Eds., "The Insect Viruses” New York, Plenum Press, pp. 301-329; van Regenmortel et al., 2000, In: "Virus Taxonomy: The Classification and Nomenclature of Viruses. The Seventh Report of the International Committee on Taxonomy of Viruses” Academic Press, San Diego).
- Genomes of a number of these viruses have now been sequenced. These include CrPV (GenBank accession number AF218039), DCV (AF014388), acute bee paralysis virus (ABPV, NC002548); black queen cell virus (BQCV, AF183905) and SBV (AF092924) of honeybees; RhPV (AF022937), PSIV (AB006531), TrV (AF178440), HiPV (AB017037); IFV (AB000906) and TSV of shrimp (AF277675).
- CrPV GenBank accession number AF218039
- DCV AF014388
- ACBPV acute bee paralysis virus
- BQCV black queen cell virus
- SBV AF092924
- RhPV AF022937
- PSIV AB006531
- TrV AF178440
- HiPV AB017037
- IFV IFV
- TSV AF277675
- the invention is a composition comprising nucleotide sequence of an insect picomavirus (IPV) such as the Taura syndrome virus (TSV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), or white spot syndrome virus (WSSV), and one or more cistronic elements (e.g., a shrimp gene sequence) operably linked there with.
- IPV insect picomavirus
- TSV Taura syndrome virus
- IHHNV infectious hypodermal and hematopoietic necrosis virus
- WSSV white spot syndrome virus
- the invention provides methods of protection of animals, in particular crustaceans from disease and manipulation of animals, specifically crustaceans, in vitro and in vivo.
- the invention provides tools critical for the production of an immortalized shrimp cell line.
- the following examples describe some aspects of the invention and are used for exemplification purposes only and are not intended to limit the scope of the invention in any way.
- IVS internal ribosomal entry site
- Insect picornavirases with dicistronic genomes have two unique features. First, no sub-genomic RNA is produced for translation of the capsid proteins. Second, the coat protein cistron appears to lack an initiating methionine suggesting that the coat protein is translated through internal initiation mediated by an internal ribosomal entry site (IRES).
- IRES elements have been identified in the intergenic region of CrPV and PSIV (Wilson et al., 2000, Mol. Cell. Biol. 20:4990-4999; Sasaki et al., 1999, J. Virol. 73:1219-1226), and homologous sequences have been identified in TSV (Mari et al., 2002, J. Gen.
- nucleotide difference in the intergenic region among insect picoma-like viruses was calculated using the computer program MEGA (Kumar et al., 2001, Molecular Evolutionary Genetic Analysis (MEGA) software, version 2.0, Institute of Molecular Evolutionary Genetics, Arizona State University, Arizona, USA) (Table 1).
- MEGA Molecular Evolutionary Genetic Analysis
- the program calculates the p-distance (nucleotide), which is defined as the proportion (p) of nucleotide sites at which the two sequences compared are different. It is obtained by dividing the number of nucleotide differences by the total number of nucleotides compared.
- nucleotide residues that constitute the stem loop structure right before the ORF2 is indicated in Figure 2C.
- This region in TSV can form secondary structures like other insect picoma-like viruses, and initiate IRES-mediated cap-independent translation of TSV ORF2 that encodes capsid proteins.
- IRES-mediated translation of the coat proteins in insect picorna-like viruses with dicistronic genome therefore, provides a mechanistic explanation for the abundance of structural compared to non-structural proteins in insect picoma-like virus infected cells.
- Insect picoma-like viruses with dicistronic genomes encode two distinct polyproteins: non-structural proteins encoded by the upstream ORF and the structural protein encoded by the downstream ORF. Translation of the two ORFs seems to be independently controlled.
- Example 2 Genome organization of IHHNV
- the IHHNV genome contains a single stranded linear DNA of 4.1 kb (Bonami et al., 1990). Almost the entire IHHNV genome except the 5'-and 3'-terminal ends has been sequenced (Shike et al., 2000, Virology, 277:167-177).
- ORFs open reading frames
- Fig. 3 The middle and the right ORFs are present in the plus 3 frame, whereas the left ORF is present in the plus 3 frame.
- the middle ORF completely overlaps with the left ORF, whereas the right ORF partially overlaps with the left ORF.
- the left, middle and the right ORFs have potential coding capacities of 666 amino acids (75.77 kDa), 363 aa (42.11 kDa) and 329 aa (37.48 kDa), respectively.
- the left ORF most likely encodes for a major non-structural protein (NS-1), which contains replication initiation motifs, NTP -binding, and helicase domains.
- the right ORF encodes the capsid protein.
- the aa sequence of middle ORF does not show any similarity with database entries, and therefore, the putative function of this ORF is unknown. There are two potential promoters identified in IHHNV genome.
- IHHNV is closely related to densoviruses of the genus Brevidensovirus in the family Parvoviridae (Shike et al., 2000, Virology, 277:167-177).
- Figure 3 A schematic representation of the genome organization of IHHNV genome is shown in Figure 3.
- IHHNP61F 5'-GGTAC CTCCA GCTGA TGGTA AAGCT-3' (SEQ ID NO: 19; nucleotide position 2346-2371 in AF273215) and IHHNP61R: 5'-TTCGT ATTCT TGGAA GAGTC CTAG3" (SEQ ID NO: 20; nucleotide position 2488 in AF273215).
- the reaction mixture for the PCR included IX Promega thermophilic DNA polymerase buffer, 2.0 millimolar MgC_2, 0.4 micromolar dNTPs, 0.8 micromolar of forward and reverse primer, 7.5 units of Promega Taq DNA polymerase, and 100 nano grams of total genomic DNA isolated from IHHNV-infected shrimp in a 25 microliter reaction volume.
- the thermal profile for the PCR was 95 degrees Celsius for 5 minutes followed by 40 cycles of 95 degrees Celsius 30 seconds, 52 degrees Celsius 30 seconds, and 72 degrees Celsius 1 minute before cooling at 4 degrees Celsius.
- PCR amplified DNA was run in a 1.5% agarose gel containing ethidium bromide using Tris-acetate EDTA buffer and photographed. A photograph of the agarose gel is shown in Figure 4.
- the amplified DNA was gel purified using Qiagen gel purification kit (Qiagen, California), and sequenced using IHHNVP61F primer.
- the nucleotide sequence of the amplified DNA showed 100% similarity with the GenBank database entry, AF273215.
- the genome of WSSV contains a circular double-stranded (ds) DNA of about 300 kb in size. So far three geographic isolates of WSSV have been completely sequenced including a Thai-isolate (292,967 bases, GenBank accession number AF369029, van Hulten et al., 2001, Virology 286:7-22), a Chinese isolate (305,307 bases, Yang et al., 2001, J. Virol. 75:11811-11820; GenBank accession number AF332093), and an isolate from Taiwan (307,287 bases, GenBank accession number AF440570). A representative genetic map of one of the isolates, Thai-isolate, is shown in Figure 5.
- the three isolates show an overall nucleotide identity of 99.32% (Marks et al., 2004, Arch. Virol. 149:673-697).
- ORFs open reading frames
- the size differences among the isolates are due to a 13210 bp deletion in the Thai-isolate and a 1168 bp deletion in the WSSV Chinese isolate compared to the WSSV Taiwan isolate (Marks et al., 2004, Arch. Virol. 149:673-697).
- WSV is morphologically similar to insect baculoviras.
- phylogenetic analysis of ribonucleotide reductase and protein kinase genes revealed that WSV does not share a common ancestor with baculo viruses (van Hulten et al., 2000, J. Gen. Virol. 81:307- 316; van Hulten et al, 2001b, Viras Genes 22:201-207).
- WSSV Due to its limited sequence similarity with other virases sequenced so far, WSSV has been placed in a new viras family, Nimaviridae, genus Whispovirus, as described in the art, for example in the NCBI's publicly-available web site relating to viral taxonomy.
- the WSSV Taiwan isolate transposase gene shows 100% similarity with the homologous transposable elements of prokaryotic and eukaryotic origin.
- the regions upstream and the downstream of the ORF contains inverted repeats, a common feature of transposable elements.
- At the site of insertion of the transposable element in the WSSV genome there has been a duplication of the WSSV sequence.
- the similarity of WSSV transposase with the homologous sequence in the GenBank database, and the genetic map of the transposase gene in WSSV genome are shown in Figures 6 and 7.
- Transposable elements have been identified from a wide variety of species including bacteria, plants, invertebrates, and vertebrates, and they constitute a considerable portion of the genetic makeup of a species.
- TEs make up 3% of yeast genome (Kim et al., 1998), 10-15% of Drosophila sp. genome (SanMiguel et al., 1996, Science 274:765-768), and about 50% of maize genome (Pimpinelli et al., 1995).
- TEs constitute 43% of the genome representing 4.3 million TEs (Smit, 1999, Curr. Opin. Genet. Dev.
- Penelope elements in Drosophila has shown to be associated with a syndrome of aberrant traits collectively known as dysgenesis (Kidwell et al., 1997, Proc. Natl. Acad. Sci. USA 94:7704-7711).
- Intact Penelope elements of Drosophila encode a reverse transcriptase and an endonuclease of the UvrC type, which may play a role in Penelope integration (Lankenau et al., 1997, Proc. Natl. Acad. Sci. USA 94:196-201).
- Pyatkov et al. 2002, Proc. Natl. Acad. Sci.
- Penelope element as a transformation vector for germ-line transformation of Drosophila. Penelope was found to be actively transcribed, and undergoes massive copy number increase only in transformed lines where transformation was performed using a full-length copy as opposed to a truncated copy of the Penelope element.
- IHHNV P61 promoter was amplified from IHHNV-infected shrimp using genomic DNA as template for the PCR
- the IHHNV P61 promoter is cloned into a TOPO vector following manufacturer's protocol (Invitrogen, Inc., California).
- the cDNA amplicon is cloned into TOPO vector (Invitrogen, Inc,
- the primer sequences for amplifying the TSV intergenic region are: Forward 5'-TAGCA
- ACGCA AG-3' (SEQ ID NO: 22).
- the insert for the TSV intergenic region is digested from the recombinant clone with appropriate restriction enzyme, run in a 1.5% agarose gel following standard protocol (Sambrook et al., 1989, "Molecular Cloning: A laboratory manual” Ed. 2, Cold Spring Harbor Press, Cold Spring Harbor) and gel purified using the Qiagen gel purification kit (Qiagen, California).
- the TSV intergenic region is cloned downstream of the IHHNV promoter.
- the plasmid DNA from a recombinant clone containing the IHHNV promoter is digested with enzymes compatible with the TSV intergenic region insert and ligated using
- the recombinant clones containing the IHHNV P61 promoter and the TSV intergenic region are identified by colony PCR using IHHNVP61F primer and a vector derived primer. Clones that contain appropriate orientation (see Fig. 9A) are taken for subsequent sub-cloning into a pDsRed vector (BD Bioscience, California). The plasmid DNA containing the IHHNV P61 promoter and the TSV intergenic region are digested with appropriate enzyme, and cloned upstream of selectable marker DsRed in pDsRed vector (BDClontech). The plasmid DNA of the recombinant clones is sequenced to confirm the identity of the transformants. A schematic representation of shrimp transient vector is presented in Fig. 9 A.
- Plasmid DNA from recombinant shrimp expression vector is injected into the tail muscle of juvenile shrimp (about 1 gram) and the activity of the reporter gene (DsRed) is assayed either directly using a transilluminator and direct observation of the shrimp or by excision of the area injected and then extraction of soluble protein and analysis on a fluorescent microplate reader (Matz et al, 1999, Nat. Biotechnol. 17:969-973).
- DsRed reporter gene
- Tissue is homogenized with a tissue homogenizer in 50 millimolar Tris (pH 7.0) buffer plus proteinase inhibitor (1 millimolar phenylmethylsulfonyl fluoride, 5 millimolar benzamidine). The soluble portion is recovered after a brief centrifugation. This is analyzed for total protein by the Lowry method and aliquotted into black Nunc microplates in serial dilution. Fluorescence is read on SpectraFluor Plus (Tecan) reader at 485 nm excitation and 535 emissions. Controls are run with standard DsRed protein either made internally or obtained commercially.
- Plasmid DNA from recombinant shrimp expression vector is injected to transfect primary cell culture of shrimp using lipofecting agent. Cells transfected with the plasmid
- DNA of non-recombinant clone serve as a control.
- the activity of the reporter gene (DsRed) is assayed at 24, 48, 72 hours post transfection following standard protocol (see Example 8).
- RT-PCR is performed using DNase treated total RNA from Penaeus vannamei shrimp to amplify a 500 bp amplicon using shrimp Penelope primers.
- the primers are designed based on the partial sequence of shrimp Penelope element available in the GenBank database (accession number AF077579).
- the RT-PCR protocol for amplifying the Penelope element is same as described for amplifying other shrimp genes like beta-actin (Dhar et al., 2002).
- the amplified cDNA is run in a 1.5% agarose gel following standard protocol (Sambrook et al., 1989, "Molecular Cloning: A laboratory manual” Ed.
- the 500 bp partial clone of shrimp Penelope element is used as a probe to pull out a full-length clone of Penelope element from a shrimp (Penaeus vannamei) cDNA library (Dhar et al, 2004, "Comparison of expression profiles of healthy and white spot syndrome virus (WSSV) infected shrimp (Penaeus vannamei) by expressed sequence tag (EST) analysis," Presented at the World Aquaculture Society meeting,shrimp Genomics Section, March 1-5, 2004, Honolulu, HI).
- WSSV healthy and white spot syndrome virus
- EST expressed sequence tag
- 5'- RACE technique is employed to clone the 5 '-terminal sequences following protocol used to clone the 5'-terrninal sequence of other shrimp gene (Roux et al., 2002, J. Virol. 76:7140- 7149). Recombinant clones are sequenced to confirm the identity of the clone.
- IHHNV P61 promoter (Fig. 4) is cloned into TOPO vector following manufacturer's recommendations (Invitrogen, Inc., California).
- the entire shrimp Penelope element ORF is amplified from the recombinant clone by PCR.
- the primers for the PCR are based on the sequence of the Penelope element ORF.
- the primer sequences contain unique restriction enzyme sites that are need to clone it downstream of IHHNVP61 promoter.
- Amplified cDNA is digested with those enzymes before cloning downstream of _HHNVP61 promoter in TOPO vector.
- TSV intergenic region between ORF1 and ORF2 is amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) following published protocol (Dhar et al., 2002, J. Virol. Meth. 104:69-82).
- RT-PCR reverse transcriptase-polymerase chain reaction
- the cDNA amplicon is cloned into TOPO vector (Invitrogen, Inc, California) and recombinant clones are sequenced to confirm the identity of the cDNA.
- the primer sequences for amplifying the TSV intergenic region are: Forward: 5'-TAGCACCACC CGATCGTAAA C-3' (SEQ ID NO: 23), Reverse 5'- TAATTAAGTC CCACCACGCA AG-3' (SEQ ID NO: 24).
- the insert for TSV IRES is digested from the recombinant clone with appropriate restriction enzyme, run in a 1.5% agarose gel following standard protocol (Sambrook et al., 1989, "Molecular Cloning: A laboratory manual” Ed. 2, Cold Spring Harbor Press, Cold Spring Harbor) and gel purified using Qiagen gel purification kit (Qiagen, California).
- the TSV intergenic region is sub-cloned upstream of DsRed open reading frame in pDsRed vector (BD Bioscience, California).
- the recombinant clones containing the TSV intergenic region is sequenced to confirm the orientation and identity of the clones.
- the TSV IRES-pDsRed plasmid is digested with two enzymes that cut upstream of the TSV IRES element. Similar enzymes are used to cut out the IHHNVP61 -Penelope construct.
- the IHHNVP61-PeneZope insert is gel purified using Qiagen gel purification kit before cloning upstream of the TSV intergenic region in pDsRed-TSV Intergenic construct.
- IHHNV P61 promoter (Fig. 4) is cloned into a TOPO vector following manufacturer's recommendations (Invitrogen, Inc., California).
- the entire shrimp WSSV transposase ORF is amplified by RT-PCR using DNase-treated total RNA from WSSV-infected shrimp tail muscle.
- the primers for the PCR are based on the sequence of the WSSV transposase ORF (GenBank accession number AF440570). The primer sequences contain unique restriction enzyme sites that are needed to clone it downstream of IHHNVP61 promoter.
- Amplified cDNA is digested with those enzymes before cloning downstream of IHHNVP61 promoter in TOPO vector. Ligation and transformation are done following standard protocol (Sambrook et al, 1989, "Molecular Cloning: A laboratory manual” Ed. 2, Cold Spring Harbor Press, Cold Spring Harbor). Recombinant clones are sequenced to confirm the identity of the WSSV transposases clones.
- TSV intergenic region (including the TSV IRES) between ORFl and ORF2 is amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) following published protocol (Dhar et al., 2002, J. Virol. Meth. 104:69-82).
- RT-PCR reverse transcriptase-polymerase chain reaction
- the cDNA amplicon is cloned into a TOPO vector (Invitrogen, Inc, California) and recombinant clones are sequenced to confirm the identity of the cDNA.
- the primer sequences for amplifying the TSV intergenic region are: Forward: 5'-TAGCACCACC CGATCGTAAA C-3' (SEQ ID NO: 25), Reverse 5'- TAATTAAGTC CCACCACGCA AG-3' (SEQ ID NO: 26).
- the insert for TSV IRES is digested from the recombinant clone with appropriate restriction enzyme, run in a 1.5% agarose gel following standard protocol (Sambrook et al., 1989, "Molecular Cloning: A laboratory manual” Ed. 2, Cold Spring Harbor Press, Cold Spring Harbor) and gel purified using Qiagen gel purification kit (Qiagen, California).
- the TSV intergenic region is sub-cloned upstream of DsRed open reading frame in pDsRed vector (BD Bioscience, California).
- the recombinant clones containing the TSV intergenic region are sequenced to confirm the orientation and identity of the clones.
- the TSV IRES-pDsRed plasmid is digested with two enzymes that cut upstream of the TSV IRES element. Similar enzymes are used to cut out the IHHNVP61-WSSV transposase construct.
- the 1HHNVP61-WSSV transposase is gel purified using Qiagen gel purification kit before cloning upstream of the TSV intergenic region in pDsRed-TSV Intergenic construct. All enzyme digestion, ligation and transformation are done following standard protocol (Sambrook et al., 1989, "Molecular Cloning: A laboratory manual” Ed. 2, Cold Spring Harbor Press, Cold Spring Harbor). Recombinant clones are sequenced to confirm the orientation and identity of the inserts.
- inverted terminal repeat elements of WSSV are amplified by RT- PCR and cloned upstream of IHHNVP61 promoter and downstream of the selectable maker gene (in this case DsRed).
- DsRed selectable maker gene
- Plasmid DNA from a recombinant shrimp transfection vector is used to transfect primary cell culture of shrimp using a lipofecting agent.
- Shrimp primary cell culture for hemocytes is made following a published protocol (e.g., Itami et al., 1999, Meth. Cell Sci. 21 : 237-244).
- Shrimp hemocyte primary culture is transfected with the plasmid DNA of shrimp transfection vector described in Example 11, Fig. 9B.
- the IHHNVP61 or P2 promoters driving Shrimp Penelope full-length ORF serve as a control.
- Adherent shrimp cells produced as in Example 13 are utilized for the diagnosis of viral disease by growth of the cells in a microplate. These cells are then exposed to shrimp tissue to be analyzed that has been macerated in a sterile tube and serially diluted using standard techniques. As infection proceeds, the fluorescence of the DsRed is monitored in a SpectraFluor Plus plate reader as described in Example 8. Cell mortality due to viral infection is reflected in a decrease in DsRed emission and viral titer can be determined rapidly versus a standard curve run simultaneously.
- the IHHNV p61 promoter element (GenBank accession # AF273215) was excised from a previously constructed and verified clone displayed in Figure 11 using
- the targeted fragment was PCR amplified using the forward primer p61
- This amplicon was then digested with S ⁇ cl and Nhel restriction enzymes (underlined in primer sequences) for insertion.
- the digested amplicon was gel purified using methods described for MiniElute Gel Extraction Kit in the manufacturer's instructions (Qiagen,
- the purified fragment was then inserted into a pGL3-Basic reporter vector containing a modified coding region for firefly (Photinus pyralis) luciferase (Promega Corp.
- the p2 promoter element from IHHNV was synthesized using methods described previously (Smith et al., 2003, Proc. Natl. Acad. Sci. USA 100:15440-15445) at a commercial facility. Briefly, the synthesized fragment was inserted into the general use cloning vector pUC19 (GenBank accession # L09137). That plasmid was then sequence verified at the same location. Using the synthesized S ⁇ cl and Nhel sequences for excision, the p2 fragment was removed and inserted into the pGL3-Enhancer vector mentioned above. Replicate clones were verified as above. This produced the construct shown in Figure 14.
- GenBank accession # AF277675 GenBank accession # AF277675 was excised from a previously constructed and verified clone. Briefly, the targeted fragment was PCR amplified using the forward primer TSV
- IGR SACF (5'-GAT CGA GCT CTA GCA CCA CCC GAT CGT AAA C-3'; SEQ ID NO:
- TSV IGR BAMR (5'-GAT CGG ATC CTA ATT AAG TCC CAC CAC-3'; SEQ ID NO: 1
- TSV-IGR 2 F 5'-ATA CTC GAG AAC TAA TAG CAC CAC CCG-3';
- TSV-IGR 2 R (5*-TCC AAG CTT TTG TTG TAT CAA AAT TAT -
- Example 17 Transient expression of a marker protein in shrimp.
- Shrimp Handling [0148] Naive juvenile shrimp (Penaeus vannamei) free of specific pathogens were obtained from an outside source (Penaeu (Litopenaeus) vannamei (Kona line), about 1 gram, obtained from The Oceanic Institute. Waimanalo, HI).
- SPF Specific Pathogen-Free shrimp were free of those pathogens cited in Lightner (2003, "Exclusion of Specific Pathogens for Disease Prevention in a Penaeid Shrimp Biosecurity Program," In “Biosecurity in Aquaculture Production Systems: Exclusion of Pathogens and Other Undesirables” The World Aquaculture Society, Baton Rouge, LA).
- the animals were held in artificial seawater at about 26 degrees Celsius containing standard sea salt components (INSTANT OCEAN® Cat# SS1-160P, Aquarium Systems Inc., Mentor, OH) until use.
- Shrimp Grower feed (2.4 millimeter 3/32, Cat# SI-35, Zeigler Bros., Inc. Gardners, PA) was given at regular intervals with the amount being about 3% of the total body weight.
- a standard curve using recombinant luciferase (QUANTILUM® recombinant luciferase.
- Source North American firefly, Photinus pyralis; Cat# E170B, Promega Corp. Madison, WI) was constructed beginning at a concentration of 1 femtogram per microliter and serially diluted 2-fold to 15.6 attograms per microliter. Duplicate wells were averaged together for producing the graph in Figure 17.
- Tissue lysate samples were read in triplicate wells. These technical replicate values were averaged together to form the chart in the data section below. Biological replicate values lying 1 standard deviation (SD) outside of the mean of the entire group were excluded. Standards were run to provide a plot of the standard curve values, shown in Figure 17.
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Plant Pathology (AREA)
- Environmental Sciences (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Biodiversity & Conservation Biology (AREA)
- Animal Husbandry (AREA)
- Animal Behavior & Ethology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
Claims
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US11/587,770 US7973148B2 (en) | 2004-04-15 | 2005-04-15 | Crustacean expression vector |
EP05740192A EP1756276A4 (en) | 2004-04-15 | 2005-04-15 | Crustacean expression vector |
US13/176,026 US20110269225A1 (en) | 2004-04-15 | 2011-07-05 | Crustacean expression vector |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US56245904P | 2004-04-15 | 2004-04-15 | |
US60/562,459 | 2004-04-15 |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US13/176,026 Continuation US20110269225A1 (en) | 2004-04-15 | 2011-07-05 | Crustacean expression vector |
Publications (3)
Publication Number | Publication Date |
---|---|
WO2005102041A2 true WO2005102041A2 (en) | 2005-11-03 |
WO2005102041A3 WO2005102041A3 (en) | 2006-03-30 |
WO2005102041B1 WO2005102041B1 (en) | 2006-11-09 |
Family
ID=35197461
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2005/012979 WO2005102041A2 (en) | 2004-04-15 | 2005-04-15 | Crustacean expression vector |
Country Status (2)
Country | Link |
---|---|
EP (1) | EP1756276A4 (en) |
WO (1) | WO2005102041A2 (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2014186334A1 (en) * | 2013-05-15 | 2014-11-20 | Robert Kruse | Intracellular translation of circular rna |
WO2020023595A1 (en) * | 2018-07-24 | 2020-01-30 | Mayo Foundation For Medical Education And Research | Circularized engineered rna and methods |
CN113302282A (en) * | 2019-01-15 | 2021-08-24 | 鲜可肉品私人公司 | Isolation and culture of muscle cells and fat cells from crustaceans |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB0019745D0 (en) * | 2000-08-10 | 2000-09-27 | Univ Surrey | Gene expression element |
JP2002306168A (en) * | 2001-01-25 | 2002-10-22 | National Institute Of Agrobiological Sciences | New translation activity-promoting higher structure |
US20060242719A1 (en) * | 2001-12-04 | 2006-10-26 | Sun Piera S | Nucleotide sequences of shrimp beta-actin and actin promoters and their use in gentic transformation technology |
-
2005
- 2005-04-15 WO PCT/US2005/012979 patent/WO2005102041A2/en active Application Filing
- 2005-04-15 EP EP05740192A patent/EP1756276A4/en not_active Withdrawn
Non-Patent Citations (1)
Title |
---|
See references of EP1756276A4 * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2014186334A1 (en) * | 2013-05-15 | 2014-11-20 | Robert Kruse | Intracellular translation of circular rna |
US9822378B2 (en) | 2013-05-15 | 2017-11-21 | Ribokine, Llc | Intracellular translation of circular RNA |
WO2020023595A1 (en) * | 2018-07-24 | 2020-01-30 | Mayo Foundation For Medical Education And Research | Circularized engineered rna and methods |
CN113302282A (en) * | 2019-01-15 | 2021-08-24 | 鲜可肉品私人公司 | Isolation and culture of muscle cells and fat cells from crustaceans |
Also Published As
Publication number | Publication date |
---|---|
WO2005102041A3 (en) | 2006-03-30 |
EP1756276A2 (en) | 2007-02-28 |
WO2005102041B1 (en) | 2006-11-09 |
EP1756276A4 (en) | 2008-10-29 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US7973148B2 (en) | Crustacean expression vector | |
Flegel | Major viral diseases of the black tiger prawn (Penaeus monodon) in Thailand | |
Sivasubbu et al. | Gene-breaking transposon mutagenesis reveals an essential role for histone H2afza in zebrafish larval development | |
Reichwald et al. | High tandem repeat content in the genome of the short-lived annual fish Nothobranchius furzeri: a new vertebrate model for aging research | |
Chiu et al. | Transgenic mice that express Cre recombinase in osteoclasts | |
Collet et al. | Engineered cell lines for fish health research | |
CN110484549B (en) | Genome targeted modification method | |
Jiang et al. | Characterization and prevalence of a novel white spot syndrome viral genotype in naturally infected wild crayfish, Procambarus clarkii, in Shanghai, China | |
Dalla Benetta et al. | Engineered antiviral sensor targets infected mosquitoes | |
Edgerton | Susceptibility of the Australian freshwater crayfish Cherax destructor albidus to white spot syndrome virus (WSSV) | |
EP1756276A2 (en) | Crustacean expression vector | |
Jeong et al. | Asymptomatic iridovirus infection in various marine fishes detected by a 2-step PCR method | |
Yan et al. | Characterization of the Drosophila suzukii β2-tubulin gene and the utilization of its promoter to monitor sex separation and insemination | |
JP2008063302A (en) | Oral vaccine for white spot disease of penaeus | |
Zieler et al. | Intron‐dependent stimulation of marker gene expression in cultured insect cells | |
Laha et al. | pido, a non-long terminal repeat retrotransposon of the chicken repeat 1 family from the genome of the Oriental blood fluke, Schistosoma japonicum | |
Zhang et al. | Extrachromosomal transposition of the transposable element Minos in embryos of the cricket Gryllus bimaculatus | |
US20060242719A1 (en) | Nucleotide sequences of shrimp beta-actin and actin promoters and their use in gentic transformation technology | |
CN114058618B (en) | Application of glutamate dehydrogenase as target in pest control | |
EP1083788B1 (en) | Transgenic fish carrying a bacteriophage-derived transgene construct for mutation detection | |
Gangnonngiw et al. | A novel ssDNA Bidnavirus in the giant freshwater prawn Macrobrachium rosenbergii | |
NO20211349A1 (en) | Sterile fish | |
US7129083B1 (en) | PiggyBac transformation system | |
Rice | Investigating pathogenicity of Spring Viraemia of Carp virus (SVCV) and the development of diagnostic tools | |
RU2798051C2 (en) | New fish totivirus |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A2 Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KM KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SM SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A2 Designated state(s): BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
NENP | Non-entry into the national phase in: |
Ref country code: DE |
|
WWW | Wipo information: withdrawn in national office |
Country of ref document: DE |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2005740192 Country of ref document: EP |
|
WWP | Wipo information: published in national office |
Ref document number: 2005740192 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 11587770 Country of ref document: US |
|
WWP | Wipo information: published in national office |
Ref document number: 11587770 Country of ref document: US |