Ryszard Slomski, Daniel Lipinski, Marlena Szalata, Joanna...
Transcript of Ryszard Slomski, Daniel Lipinski, Marlena Szalata, Joanna...
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Bioreactors and transgenic animals
Ryszard Slomski, Daniel Lipinski, Marlena Szalata, Joanna Zeyland, Jacek Jura, Zdzislaw Smorag
Poznan University of Life Sciences, Poznan, PolandInstitute of Human Genetics, PAS, Poznan, Poland
National Research Institute of Animal Production, Cracow, Poland
TRENDS IN SCIENCE AND TECHNOLOGYRELEVANT TO THE BIOLOGICAL AND TOXIN WEAPONS
CONVENTION
31 October─3 November 2010
Institute of Biophysics, Chinese Academy of SciencesBeijing, China
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Bacteria Yeast Fungi Plants Cell cultures
Animals
Speed ++++++ ++++ +++++ ++ +++ +Total costs ++++ ++ +++ ++++++ + +++++Post-translational modifications
+ ++ +++ ++++ ++++++ +++++
Up-scaling ++++ ++ +++ ++++++ + +++++Regulations +++++ ++++ +++ ++ ++++++ +
BioreactorsProduction methods
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1. Selection of valuable protein for production.2. Selection of targeted site of transgenesis.3. Selection of organism for transgenesis
Yield of production per year/required volume Processing potentialsCulturing/breeding problemsUtilization of the recombinant productTime needed to manufacture product
BioreactorsSpecific aims
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BioreactorsFrom idea to product
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Purification under native (lanes 1-8) or denaturing conditions (lanes 9-15). Lanes 1,2,3,9,10,11,12, different clones with FeldI chain 1 protein, lanes 4,5,6,12,13,14, different clones with FeldI chain 2, lanes 7,8,15,controls. Lanes 12,13,14 expression 106 amino acid peptide in E.coli.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Screening of bacterial culturesoverexpressing gene construct
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Left site: overexpression of FeldI chain 1 protein. Lane 1, control; lane 2, overexpression; lane 3, proteins not bound to column; lanes 4-7, fractions of purified protein; lane 8, marker (16.9, 14.4, 8.2 Da).
Right site: overexpression of FeldI chain 2 protein. Lane 1, control; lane 2, overexpression; lane 3, proteins not bound to column; lanes 4-7, fractions of purified protein; lane 8, marker (16.9, 14.4, 8.2 Da).
FeldI chain 1 FeldI chain 21 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8
Purification of FeldI proteins overexpressed in E.coli by metal affinity chromatography
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Purification of recombinant Glu by metal affinity chromatography. Lane 1, cell lysate; lane 2, unbound protein; lanes 3-9, elutions; lane 10, weight marker (14.4-116 kDa). Recombinant Glu peptide is indicated by an arrow.
1 2 3 4 5 6 7 8 9 10
Purification of recombinant Cat by metal affinity chromatography. Lane 1, cell lysate; lane 2, unbound protein; lanes 3-8, elutions; lane 9, weight marker (14.4-116 kDa). Recombinant Cat peptide is indicated by an arrow.
1 2 3 4 5 6 7 8 9
Overexpression and purification of recombinant Glu and Cat domain of S. mutans
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Blood
Urine
Seminal plasma
Egg white
Silk worm cocoon
Milk
Animal bioreactorsAnimal systems for production
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Growth factors Hormones Antiviral proteins Lipocortins Lipotropins Interleukins Interferons Stimulating factors Kinases
Transmembrane regulators Immunoglobulins Milk lipases Cell surface proteins Human pancreatic enzymes Enkephalins Silk proteins Spider silk proteins
Animal bioreactorsExemplary categories of polypeptides
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Silkworm (Bombyx mori) - natural silk manufacturer for textile industryCocoon – potential source of high amounts of
recombinant proteinsShort time of generationProduction of vaccines - cholera toxin B subunit fusion
protein linked with human insulin B chain peptide at levels up to 0.97 g/l of hemolymph)
Animal bioreactorsSilkworm larvae
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A. EmbryoB. BrainC. Brain sectionD. Brain sectionE. Silk producing glandF. Silk producing gland sectionG. GonadH. Gastrointestinal tract and
body cover
J. Thomas, 2003
Expression of LacZ gene of pBRJZ vector in embryonic tissues of Bombyx morisubjected to transgenesis by biollistic method.
Animal bioreactorsExpression analysis
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Value addition (increase one of the casein components in milk for production of cheese or yogurt)Milk of higher nutrient content (alteration of milk
composition has the potential to enhance the production of certain proteins and/or growth factors that are deficient in milk)Production of proteins affecting human and animal
health (human butyrylcholinesterase, mastitis)
Animal bioreactorsMammary gland
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Rabbit (human IGF-1, human tissue plasminogen activator, erythropoietin, α-glucosidase, factor NGF-β, protein C, human growth hormone, rotavirus inner core proteins, human factor VIII, human alpha 1,3 fucosyltransferase)Goat (human lactoferrin, E2–CSFV vaccine, human tissue
plasminogen activator, human antithrombin III, human monoclonal antibodies, growth hormone)Sheep (human factor VIII, human factor IX, human α-1-
antitrypsin, fibrinogen)Pigs (protein C, human factor VIII)Cows (human lactoferrin, human erythropoietin, human
serum albumin)
Animal bioreactorsMammary gland
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Promoter and functional gene
Detection system
Purification system
Animal bioreactorsDesigning of gene construct
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Animal bioreactorsWAP:FUC rabbits
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Animal Pregnancy(months)
Maturation(months)
Amount of milk per lactation (l)
Months from microinjection to milk production
Mouse 0.75 1 0.0015 3-6Rabbit 1 5-6 1-1.5 7-8
Pig 4 7-8 200-400 15-16Sheep 5 6-8 200-400 16-18Goat 5 6-8 600-800 16-18Cow 9 15 8000 30-33
Animal bioreactorsComparison of the production of milk using different
transgenic animal species
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High efficiency of expressionProper posttranslational modificationsLow maintaining costsHigh reproductivity of transgenic foundersBreeding in pathogen free environmentLack of human infecting agents
Animal bioreactors
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Transgenic rabbits 05 and 08Generation F1
Animal bioreactorsDetection & stability
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Transgenesis was confirmed by molecular and cytogenetic analysis
Homozygous transgenic female 12B
Animal bioreactorsFinal stage of transgenesis - production
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Purification of human growth hormone from milk of transgenic rabbit. Samples of milk collected from lactating females were subjected to metal affinity column chromatography (Talon).
1 2 3 4 5 6 7 8
43 kDa43 kDa
30 kDa30 kDa
17.2 kDa
12.3 kDa
Animal bioreactorsRecombinant protein purification
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Animal bioreactorsBlastomere cloning
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ATIII, goat milk, GTC BiotherapeuticsATryn®, recombinant form of human antithrombin, is the
first transgenically produced protein to be approved anywhere in the world, having recently been approved by the European Commission for the prophylactic treatment of deep vein thrombosis in patients with hereditary antithrombin deficiencies that are undergoing surgical procedures.
Animal bioreactorsATryn® - recombinant human antithrombin
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Comparison of number of patients waiting for transplants of vascularized organs with number of transplantations. The U.S. Organ Procurement and Transplantation Network and the Scientific Registry of Transplant Recipients, Annual Report 2008
XenotransplantationWaiting list
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Structure of carbohydrate ends of AB0 antigens andα1,3Gal [Gal(α1,3)Gal] epitope
XenotransplantationCell surface antigens
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Phylogenetic evolution tree of epitope α1,3Gal. Human galactosyltransferase gene (HGT2) originated before differentiation of New World Monkeys (A) or after differentiation (B)
D. Lipinski, R. SlomskiD. Lipinski, R. Slomski
XenotransplantationEpitope Gal evolution
New World MonkeysNew World Monkeys
MenMen Great ApesGreat ApesOld World MonkeysOld World Monkeys
New World MonkeysNew World MonkeyshGThGT
CattleCattlePigPig MouseMouse
ABOABO
AAMenMen Great ApesGreat Apes
Old World MonkeysOld World MonkeyshGThGT
CattleCattlePigPig MouseMouse
ABOABO
BB
CH2OHO
Gal 1
23
4
6 CH2 OH
OGal12
34
6
1,3
5 5
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1. Gene constructs for inactivation of specific genes.
2. Gene constructs for modification of specific genes.
3. Gene constructs for regulation of specific genes.
XenotransplantationSpecific aims
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1. Preparation of transcript.2. Synthesis of cDNA.3. Cloning in vectors.
XenotransplantationCloning of genes
28 S
18 S
5 S Amplification of human α1,2-fucosyltransferase (FUT) gene and human α-galactosidase (GAL) gene
Initial RNA preparations
FUT 1098 bp GAL 1290 bp
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Gene construct encoding human α1,2-fucosylotransferase under CMV promoter, competing with endogenous α1,3-galactosyltransferase for the same substrate N-acetyllactosamine.
XenotransplantationPreparation of gene construct
Restriction analysis of pCMVFUT gene construct. Vector was hydrolyzed with XbaI and BamHI enzymes (cloning sites of modified sequence encoding α1,2-fucosyltransferase in pGT-N29 vector, containing cloned CMV promoter and poly(A) of hGH gene).
α1,2-FUT
Vector
Insert
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XenotransplantationMicroinjection
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Competitive gene constructs α1,2 fucosyltransferase α galactosidase
Inactivating gene constructs Disrupted α1,3 galactosyltransferase
Regulatory gene constructs Inhibitors of complement
TG1154TG1154
XenotransplantationTransgenic pigs
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pCMVFUT gene construct and products of two PCR reactions (144 bp and 343 bp). Lanes 1-16, DNA of potentially transgenic pigs; lane 17, negative control (-DNA); lane 18 (W), positive control (CMV:Fut gene construct); lane 19 (M), size marker 267 bp and 745 bp.
XenotransplantationCMVFUT transgene detection
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FISH localization of transgene on metaphase chromosomes of transgenic pigs. On the left metaphase plate of heterozygote TG1154 boar pCMVFUT transgene on chromosome 14q28. On the right metaphase plate of homozygote 433 pig, after TG1154 boar with pCMVFUT transgene.
XenotransplantationMapping of CMVFUT transgene in F2 generation
F0 F2F2
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Stability of transgenesis.F1 offspring
11541154(TG1154)(TG1154)
162162
163163
164164
165165
166166
167167
168168
169169
170170
Negative controlNegative control(human DNA(human DNA
Negative controlNegative control(no DNA)(no DNA)
Positive controlPositive control(pCMVFut)(pCMVFut)
Marker 50Marker 50--500500
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Nucleus of WAPhGH transgenic rabbit fibroblast was analyzed using 3D FISH methods with confocal microscopy LSM 510 (analysis of xy axis).
XenotransplantationCytogenetics of transgenic animals
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RT-PCR analysis of expression of FUT transgene under control of CMV promoter in ear specimens of TG1154 boar in comparison with control pigs (1152, 1155). 400 bp long fragment of cDNA of α1,2-fucosyltransferase was amplified. Transgene was indicated by arrow. Lanes 1-3, RNA purity control: lane 1, boar TG1154; lane 2, non transgenic pig 1152; lane 3, non transgenic pig 1155; lanes 4-6, cDNA positive controls, cDNA of β-actin gene: lane 4, boar TG1154; tor 5, non transgenic pig 1152;lane 6, non transgenic pig 1155; lanes 7-9, detection of presence of cDNA of FUTtransgene: lane 7, transgenic boar TG1154; lane 8; negative control, non transgenic pig 1152; lane 9; negative control, non transgenic pig 1155; lane 10, negative control (no cDNA); lane 11, positive control (pCMVFUT plasmid); lane 12, size marker, DNA of phage hydrolyzed with HindIII and EcoRI enzymes.
XenotransplantationExpression of FUT transgene
11 2 3 2 3 44 5 6 5 6 77 8 9 10 11 128 9 10 11 12
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RT-PCR analysis of expression of FUTtransgene under control of CMV promoter in tissues of transgenic pig 166 (heart, kidney, liver, skeletal muscle and ovary) in comparison with tissues of control non transgenic pig 167. 400 bp transcript fragment of α1,2FT was amplified. Lanes 1-10, RNA purity control, β-actin genomic sequence, RNA isolated from tissues of transgenic and non transgenic pigs; lanes 12-21, cDNA positive control, tissues of transgenic and non transgenic pigs, cDNA sequence of β-actin gene (300 bp); lanes 23-27, transgenic pig 166; lane 28-32, negative control, tissues of non transgenic pig; lane 33, negative control, no cDNA; lane 34, positive control, pCMVFUT plasmid; lane 11,22,35, size marker, DNA of phage hydrolyzed with HindIII and EcoRI enzymes
XenotransplantationExpression of FUT transgene
1 2 3 4 5 6 7 8 9 10 11
12 13 14 15 16 17 18 19 20 21 22
23 24 25 26 2728 29 30 31 32 33 34 35
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Flow cytometry analysis of fibroblasts isolated from control, transgenic boar TG1154 and homozygous pig 433 after staining with UEA-1 lectin (left) (detects H antigen) and BS-IB4 lectin (right) (detects Gal epitope). The x-axis shows the fluorescence intensity, and the y-axis shows the relative cell count for unstained cells (black thin dashed-dot line), control (red thin solid line), boar TG1154 (blue thick solid line) and pig 433 (purple thick dotted line). The analysis demonstrates that increased expression of H antigen correlates with reduced expression of Gal epitope on the cell surface of transgenic pigs (boar TG1154 and pig 433).
XenotransplantationStatistical analysis of survival of transgenic cells
H antigenH antigen Gal epitopeGal epitope
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Mean values of the viability (%) of the non transgenic lines andfour transgenic lines (pCD46, pCD55 pCD59 and triple-transgenic) in the human complement-mediated cytolysis assay.
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
0% 20% 30% 40% 50%
Human serum
Viability
nontransgenictriple-transgenic
XenotransplantationStatistical analysis of survival of transgenic cells
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XenotransplantationRecently developed pig with α-galactosidase gene
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Venter "a very important philosophical step in the history of our species. We are going from reading our genetic code to the ability to write it. That gives us the hypothetical ability to do things never contemplated before".
Team of 20 top scientists, led by the H. Smith has constructed a synthetic chromosome based on the bacterium Mycoplasma genitalium, that is 381 genes long and contains 580,000 base pairs of genetic code. The synthetically reconstructed chromosome has bee named Mycoplasma laboratorium. It was then transferred into a living bacterial cell and in effect become a new life form.
Creation of artificial life
“I am creating artificial life”, declared in October 2007 US C. Venter researcher involved in the race to sequence the human DNA. He has built a synthetic chromosome and announced the creation of the first new artificial life form on Earth.
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The new life form will depend for its ability to replicate itself and metabolize on the molecular machinery of the cell into which it has been injected, and in that sense it will not be a wholly synthetic life form.
Pat Mooney, director of a Canadian bioethics organization, ETC Group: "Governments, and society in general, is way behind the ball. This is a wake-up call - what does it mean to create new life forms in a test-tube?" Craig Venter responded: “We are trying to create a new value system for life. When dealing at this scale, you cannot expect everybody to be happy."
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Thank you for your attention