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

Bacteria Yeast Fungi Plants Cell cultures

Animals

Speed ++++++ ++++ +++++ ++ +++ +Total costs ++++ ++ +++ ++++++ + +++++Post-translational modifications

+ ++ +++ ++++ ++++++ +++++

Up-scaling ++++ ++ +++ ++++++ + +++++Regulations +++++ ++++ +++ ++ ++++++ +

BioreactorsProduction methods

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

BioreactorsFrom idea to product

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.

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Screening of bacterial culturesoverexpressing gene construct

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

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.

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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.

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Overexpression and purification of recombinant Glu and Cat domain of S. mutans

Blood

Urine

Seminal plasma

Egg white

Silk worm cocoon

Milk

Animal bioreactorsAnimal systems for production

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

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

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

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

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

Promoter and functional gene

Detection system

Purification system

Animal bioreactorsDesigning of gene construct

Animal bioreactorsWAP:FUC rabbits

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

High efficiency of expressionProper posttranslational modificationsLow maintaining costsHigh reproductivity of transgenic foundersBreeding in pathogen free environmentLack of human infecting agents

Animal bioreactors

Transgenic rabbits 05 and 08Generation F1

Animal bioreactorsDetection & stability

Transgenesis was confirmed by molecular and cytogenetic analysis

Homozygous transgenic female 12B

Animal bioreactorsFinal stage of transgenesis - production

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

Animal bioreactorsBlastomere cloning

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

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

Structure of carbohydrate ends of AB0 antigens andα1,3Gal [Gal(α1,3)Gal] epitope

XenotransplantationCell surface antigens

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

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

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

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

XenotransplantationMicroinjection

Competitive gene constructs α1,2 fucosyltransferase α galactosidase

Inactivating gene constructs Disrupted α1,3 galactosyltransferase

Regulatory gene constructs Inhibitors of complement

TG1154TG1154

XenotransplantationTransgenic pigs

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

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

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

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

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

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

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12 13 14 15 16 17 18 19 20 21 22

23 24 25 26 2728 29 30 31 32 33 34 35

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

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

XenotransplantationRecently developed pig with α-galactosidase gene

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.

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."

Thank you for your attention