Conceptos y Técnicas de Biotecnología I 2011 – 2do cuatrimestre FBMC-FCEN-UBA

Transferencia de Genes a Células Animales en Cultivo Unidad de Transferencia Genética Instituto de Oncología “Ángel H. Roffo” Universidad de Buenos Aires

ORGANISMOS TRANSGÉNICOS - INVOLUCRADOS EN BIOTECNOLOGÍA •BACTERIAS (i.e.: Escherichia coli) •HONGOS (i.e.: Saccharomyces cereviseae) •CULTIVOS CELULARES (animales o vegetales) •PLANTAS ALGAS VASCULARES •ANIMALES: PECES AVES MAMÍFEROS Bovinos Caprinos Ovinos Porcinos HUMANOS: Transgénesis parcial  Terapia Génica

Transferencia de Genes a Células Animales en Cultivo PROPÓSITOS •Confirmar identidad de genes •Caracterizar oncogenes •Expresar proteínas que necesitan modificaciones posttraduccionales •Producir grandes cantidades de proteínas que naturalmente se encuentran en cantidades limitadas •Estudiar síntesis y transporte intracelular •Expresar secuencias genómicas que contienen intrones •Estudiar mecanismos de edición de genes •Analizar señales de control de transcripción y su modulación por drogas, hormonas, estado de diferenciación

Heterologous Protein Production In Eukaryotic Cells • Prokaryotic systems are generally cheaper, but… • Eukaryotic proteins produced in bacteria may be  Unstable or lack biological activity due to lack of posttranslational modifications or correct assembly  Possess unacceptable contaminants after purification

Posttranslational Modifications • • • • •

Conformation Cleavage Correct disulfide bond formation Protein disulfide isomerase Amino acid removal from initial polypeptide • O-linked or N-linked glycosylation  About 30% of eukaryotic proteins are glycosylated

Examples of O-Glycosylations

Examples of N-Glycosylations

Examples of N-Glycosylations • Initial groups can be trimmed and then expanded to create different final modifications

Examples of N-Glycosylations • Another Variation

Cultivate mammalian cells!!!! Carrel (surgeon, 1923) Aseptic techniques Carrel Flask

50-s Chemically defined media (Eagle, Earle) Consistency Sterilization Reduced chance of contamination

Growth of Animal Cells in Culture • In vitro cell culture systems enable scientists to: – study cell growth and differentiation – perform genetic manipulations to understand gene structure and function. • Culture media contains: – Serum – Salts – Glucose – Various amino acids and vitamins that the cells do not make for themselves.

Ejemplo de medio de cultivo de células de mamífero: Dulbecco Modified Eagle medium (DMEM) Vitamins

Inorganic Salts Calcium Chloride

0.2

0.2

Choline Chloride

0.004

0.004

Ferric Nitrate • 9H2O

0.0001

0.0001

Folic Acid

0.004

0.004

Magnesium Sulfate (anhydrous)

0.09767

0.09767

myo-Inositol

0.0072

0.0072

Potassium Chloride

0.4

0.4

Niacinamide

0.004

0.004

Sodium Bicarbonate

3.7

3.7

0.004

0.004

Sodium Chloride

6.4

6.4

D-Pantothenic Acid (hemicalcium)

Sodium Phosphate Monobasic (anhydrous)

0.109

0.109

Pyridoxal • HCl

—

—

Pyridoxine • HCl

0.004

0.004

Riboflavin

0.0004

0.0004

Thiamine • HCl

0.004

0.004

D-Glucose

4.5

4.5

Phenol Red • Na

0.0159

—

Pyruvic Acid • Na

0.11

—

0.584

0.584

Amino Acids L-Arginine • HCl

0.084

0.084

L-Cystine • 2HCl

—

0.0626

Glycine

0.03

0.03

L-Histidine • HCl • H2O

0.042

0.042

L-Isoleucine

0.105

0.105

L-Leucine

0.105

0.105

L-Lysine • HCl

1.46

0.146

L-Methionine

—

0.03

L-Phenylalanine

0.066

0.066

L-Serine

0.042

0.042

L-Threonine

0.095

0.095

L-Tryptophan

0.016

0.016

L-Tyrosine • 2Na •2H2O

0.10379

0.6351

L-Valine

0.094

0.094

Other

Add L-Glutamine

+ compuestos no-definidos: suero sanguíneo (5-15%), cocktail de factores de crecimiento, etc.

Serum • 0-20% Serum – – – – – –

Growth factors Transferrin (Fe) Lipids Insulin Shear protection Detoxification

Problems – Infectious agents (viruses, mycoplasm, prions)

– serum composition is poorly defined and the batches vary.

– Expensive Completely

mammalian origin free (MOF) chemically defined medias

Growth of Animal Cells in Culture • Primary cultures are the original cultures established from a tissue. • Permanent (or immortal) cell lines are embryonic stem cells or tumor cells that proliferate indefinitely in culture.

Growth curves in yeasts and mammalian cells are different

10.0

Stationary

Cx (g/l)

1.0

decline

0.1

exponential

0.0

Yeast Hybridoma

0.0

Minimum 0.0 density

Lag phase

0.0 0

50

Time (h)

100

Ejemplo: historia del desarrollo de la línea HEK-293

Graham, et al., J. Gen. Virol., 36:59-72, 1977

Dificultades suplementarias • El tiempo de división aumenta con la talla: – Las células animales necesitan condiciones de asepsia muy estrictas • Adhesión obligatoria: – Ciertas líneas de células de mamíferos necesitan un soporte para ser viables. Esto: – presenta un problema de escalado – las vuelve particularmente susceptibles a la disrupción

Ej: tapiz celular de mioblastos

Cultivo sobre microcarrioers

MODALIDAD •Transitoria: s/ Integración: Expresión 12-72 horas •Permanente: c/ Integración: Expresión 1-3 semanas

PARÁMETROS A CONSIDERAR •Tipos celulares disponibles •Expresión transitoria o permanente (estable) •Elementos de control de expresión adecuados

Common cell lines CHO

Epithelial

Chinese Hamster Ovary

HeLa

Epithelial

Human cervical carcinoma

MDCK

Epithelial

Canine Kidney

BHK

Fibroblast

Baby Hamster Kidney

Vero

Fibroblast

Monkey Kidney

WI-38

Fibroblast

Human fetal lung

3T3

Fibroblast

Mouse fibroblast

MARCADORES FENOTÍPICOS •Crecimiento en agar •Crecimiento con bajo suero •Cambios morfológicos •Rescate de la muerte de cultivos celulares primarios

MARCADORES BIOQUÍMICOS Indicadores •Anticuerpos específicos •Chloramphenicol acetyltransferase (cat) •β-galactosidase (β-gal) •Luciferase Selectores •Thymidine kinase (tk) •Xantine guanine phosphoribosyl transferase (xgprt) •Aminoglicoside phosphotransferase (apht/neor) •Dihydrofolate reductase (dhfr) •Hygromycin B phosphotransferase (hygr) Indicadores/Selectores •Green/Blue/Red fluorescence proteins (gfp/bfp/rfp)

MARCADORES BIOQUÍMICOS Indicadores •Anticuerpos específicos Ag + AbI  Ag.AbI Ag.AbI + AbII.  Ag.Ab.AbII.

: enzima, grupo fluorescente, grupo radioactivo

•Chloramphenicol acetyltransferase (cat) [14C] Chloramphenicol  Ac- [14C] Chloramphenicol  (Ac)2-[14C] Chloramphenicol

•β-galactosidase (β-gal, lac Z) lactosa  galactosa + glucosa ONPG  o-nitrofenol (amarillo) + galactosa Xgal  X (azul) + galactosa

•Luciferase Luciferina + ATP  Luciferina + ADP + h (luz)

Indicadores/Selectores •Green/Blue/Red fluorescence proteins (gfp/bfp/rfp) XFP + h1 (luz)  XFP + h2 (luz)

1 >2

Selector: FACS

Fluorescence-activated cell sorter

Reporter gene systems 1. chloramphenicol acetyl transferase (CAT) CAT is a bacterial enzyme that catalyzes the transfer of acetyl groups from acetyl-coenzyme A to the antibiotic chloramphenicol. (chloramphenicol deactivation) thin-layer chromatographic sheet Chloramphenicol is radiolabelled

For mammalian cells it is laborous and expensive. Extract protein and measure activity…

β-galactosidase (βgal) systems

luciferase (luc) systems firefly species Photinus pyralis Expressed luciferase catalyses oxidation of compounds called luciferans ( ATP-dependent process) mouse with a strain of salmonella

luciferans emit fluorescense luminometer measurement Mice are injected with LUC+ salmonellas. Sensitive digital cameras allow non-invasive detection. For GT vectors pictures look the same

Green fluorescent protein (GFP) autofluorescent protein from Pacific Northwest jellyfish Aequorea victoria GFP is an extremely stable protein of 238 amino acids with unique post-translationally created and covalently-attached chromophore from oxidised residues 65-67, Ser-Tyr-Gly

ultraviolet light causes GFP to autofluoresce In a bright green color

Jellyfish do nothing with UV, The activate GFP by aequorin (Ca++ activated, biolumuniscent helper)

Green fluorescent protein (GFP)

GFP expression is harmless for cells and animals GFP transgenic mice from Osaka University (Masaru Okabe) GFP construct could be used for construct tracking in living organism GFP labelled image of a human tumor. Vessel on the tumor surface are visible in black

MARCADORES BIOQUÍMICOS Selectores •Thymidine kinase (tk):) dT +ATP dTMP +ADP dUMP  dTTP

½ select. céls. tk-: HAT (hipoxantina, aminopterina, timidina) inhib: aminopterina

•Xantine-guanine phosphorybosil transferase (xgprt) Xantina XMP  GMP   guanina  inhib: ac. aminofenólico precursores   IMP inhib: aminpoterina  ASMP  AMP

½ selectivo p/toda cél.: AAMX (adenosina, aminopterina, ac. Aminofenólico, xantina) ASMP: ac. adenilosuccínico

•Aminoglicoside phosphotransferase (apht/neor) neomicina/kanamicina/geneticina (G418)  antibiótico fosforilado (inactivo)

•Dihydrofolate reductase (dhfr) DHFTHF ½ selectivo en céls. dhfr-: ausencia de nucleósidos ½ selectivo p/toda cél.: methotrexate (MTX)

•Hygromycin B phosphotransferase (hygr) Hygromicina B  antibiótico fosforilado (inactivo)

Gene amplification

TRANSFERENCIA GENÉTICA: METODOLOGIA

• Vectores virales • Vectores no virales Direccionamiento de vectores • Via de administración • Receptores celulares • Promotores específicos de tejido

• Vectores Virales Virus naturales (silvestres): •Crecen en todas las células •Infectan todas las células •Son patogénicos Virus terapeúticos (recombinantes): •Sólo crecen en células empaquetadoras •Infectan todas las células •No son patogénicos

Figure 1. Construction of recombinant adenoviral vectors. cDNA of interest is cloned into a shuttle vector which provides cDNA expression cassette (adenovirus ITR, E1 enhancer, adenovirus encapsidation signal, CMV promoter, and SV40 a polyadenylation signal). Homologous recombination sequences are also cloned in this vector. Adenovirus genome (e.g., pJM17 shown in the figure) and the shuttle vector containing the cDNA are cotransfected in 293 cells. Intracellular homologous recombination between the two DNAs results in a E1- recombinant genome; the numbers 0, 20, 100 represent the approximate map units. This recombinant genome is replication defective. However, in the presence of E1 proteins (provided in trans by 293 cells), the recombinant genome will replicate and form adenoviral particles.

Cytopathic Effect • Cytopathic Effect can be seen in cell monolayer • CPE is assessed at day 2, 4 and 7

Plaque Forming Units • In serial viral dilutions, areas of lysis are observed where cells are destroyed • Crystal Violet staining

Retrovirus

Adenovirus

Herpes virus

Virus Adeno asociados

8 kb

35 kb

30 kb

4,8 kb

Sólo en división activa Ex vivo o in situ

En división activa o sin división Ex vivo o in situ

En división activa o sin división Ex vivo o in situ

En división o quizás sin división Ex vivo o in situ

Estable

Transitoria

Transitoria

Posiblemente estable

Moderado

Elevado

Moderado

Moderado

Posible integración mutagénica

Reacciones inflamatorias/ inmunitarias

No

Sí

Sí

Sí

Recombinación con el hospedador

Improbable

Posible

Posible

Improbable

Recombinación con el virus parental

Imposible

Posible

Posible

Posible

VECTORES VIRALES Tamaño máximo del gen terapeútico Células objetivo

Administración Expresión del gen terapeútico Indice de expresión del gen terapeútico Riesgos

Inmunidad preexistente en el hospedador

Posible integración mutagénica

Posible integración mutagénica

• Vectores no Virales •Microinyección / Perforación (DNA desnudo) •Precipitación con fosfato de calcio •Liposomas aniónicos •Complejos DNA/lípido catiónico: lipoplex •Complejos DNA/polycatión: polyplex •Conjugados moleculares •Dendrímeros: PEI •Hydrogel •Nanoesferas Biopolímero-DNA •Complejos LPD •Inyección a presión •Electroporación •Cañón génico •Ultrasonido •Campo magnético

Microinjection

Transfection

Lipofection

Electroporation

Liposomes Why naked DNA? Lets’ wrap it in something safe to increase transfection rate Lipids – is an obvious idea !

Therapeutic drugs

Liposomes are formed by the self-assembly of phospholipid molecules in an aqueous environment. Anionic liposome

www.emc.maricopa.edu/faculty/ farabee/BIOBK/

Cationic liposomes Positively charged lipid heads

positively charged lipid droplets can interact with negatively charged DNA to wrap it up and deliver to cells Inside liposomes DNA is resistant to degradation Lipofectin, lipofectamine, lipofectase….

Lab procedure for liposome preparation

Lípidos catiónicos: Citofectinas

Complejo DNA/lípido catiónico: Lipoplex

Electron photomicrographs of lipid-DNA complexes.

Electron photomicrographs of lipid-DNA complexes. Lipid-DNA complexes were prepared at a ratio of 5:1 (w/w). PanelA shows appearance of plasmid DNA without lipid. Panels B-Fshow examples of the various types of complexes that were observed. In panelB the open arrow shows uncomplexed plasmid and the solid arrow shows plasmid complexed with lipid. Bar indicates 100 nm. DMRIE:DOPE 1:1 Zabner J et al. J. Biol. Chem. 1995;270:18997-19007

Electron photomicrographs of COS cells transfected with gold-labeled DNA complexed with lipid (DMRIE:DOPE 1:1).

Zabner J et al. J. Biol. Chem. 1995;270:18997-19007

Electron photomicrographs of COS cells transfected with gold-labeled DNA complexed with lipid. Cells were exposed to DMRIE/DOPE•DNA complexes and then removed for electron microscopy at the following times: panel A, 5 min;panel B, 30 min; panel C, 1 h;panel D, 6 h; panel E, 24 h;panel F, 24 h. Cells transfected with plasmid that had not been labeled with gold are shown inpanelF. Bar indicates 100 nm.

Protein-mediated plasmid nuclear import. Transcription factors and other nuclear proteins normally enter the nucleus through interactions between their NLSs and importin family members. However, if plasmids containing certain sequences that act as scaffolds for transcription factors and other DNA binding proteins (termed ‘DTS’, or DNA nuclear targeting sequences) are deposited into the cytoplasm during transfection, they can form complexes with these proteins, thereby attaching NLSs to the DNA. Some, but not all, of these NLSs may be in a conformation able to interact with importins for transport of the DNA– protein complex into the nucleus through the nuclear pore complex.

Methods to enhance plasmid nuclear import. A number of different approaches have been developed to promote recognition of plasmids by importin family members to increase nuclear import. These include peptide-nucleic acid clamp-conjugated NLS peptides bound to DNA, sequence-specific DNA binding proteins bound to DNA, NLS peptides covalently attached to DNA and NLS peptides electrostatically bound to DNA.

•Lipids / Polycations / DNA (LPD) Complexes LPD-I: Cationic Liposome Entraped, Polycation – Condensed DNA 

 LPD-II: Anionic Liposome Entraped, Polycation – Condensed DNA

How to make the gene expressed in the target cell? Four basic types of expression vectors : 1. Minimal promoters used to study gene regulatory elements such as enhancer elements (in the lab studies). 2. Constitutive promoters used to direct expression of gene products to produce enough target protein. 3. Cell-specific promoters used to specify expression to target cells (tissue-specific promoters in case of GT) 4. Regulated promoters used to control the on/off expression of cloned genes.

JUST TATA box and reporter Sites for constitutive transfactors

Sites for cell-specific transfactors

Sites for small ligand responsive transfactors

www.biochem.arizona.edu

Examples of often used promoters Minimal promoter

deleted Drosophila alcohol a ubiquitous low level promoter that is used to construct dehydrogenase promoter reporter genes

High activity constitutive promoters

cytomegalovirus immediate early promoter (CMV)

high level gene expression in mammalian cells

simian virus 40 early enhancer/prom. SV40

Moderately high level gene expression in mammalian cells

whey acidic protein promoter (WAP)

targeted expression of genes to mammary tissue in animals

lymphocyte-specific tyrosine kinase promoter (LCK)

targeted expression of genes to mouse thymocytes for immunological studies

mouse mammary tumor virus long terminal repeat enhancer/promoter MMTV)

steroid-regulated gene expression in mammalian cells

TET-off and TET-on systems based on tet-resistance operon of the E. coli Tn10 transposon

Regulated by doxycycline (tetracycline)

Cell-specific promoters

Regulated promoters

Generalized Eukaryotic Cloning Vector • Prokaryotic origin of replication, selectable marker • Eukaryotic origin, selectable marker • MCS with eukaryotic promoter and transcriptional terminator/polyadenylatio n signal

Mammalian Systems • Sometimes insect cells simply don’t carry out proper/necessary glycosylations • Other processing may also not occur • Mammalian cell systems are more expensive by may be required for active product

Mammalian Expression Vector

• “I” is an intron that enhances expression • Other signals similar to insect and prokaryotic vectors

Translation Control Elements

• • • • • •

K - Kozak Sequence (equiv. To rbs) S - for secretion signal peptide T – tag peptide for purification P – proteolytic cleavage sequence SC – stop codon for translation 3’UTR – proper sequences for efficient translation and mRNA stability (e.g. polyadenylation sequence)

Two Vector Expression System

•Useful for proteins of two different polypeptides

Two Gene Expression Vector

Bicistronic Expression Vector

IRES from mammalian virus

•Gives more uniform level of expression of two genes

Tetracycline-responsible systems Manfred Gossen and Hermann Bujard control the expression of genes that have been cloned downstream of a promoter containing tetracycline receptor (TetR) binding sites. VP is derived from the herpes simplex virus VP16 protein. VP – RNA pol interacting part TET-VP producing vector TET-OFF system TetR - tet binding part Gene of interest expressing vector The "Tet-off" system is repressed in the presence of the doxycycline

www.biochem.arizona.edu

"Tet-on" system is activated in the presence of doxycycline the DNA binding domain of the Tet-on regulator (rTetR) contains mutations

RNA-pol repressor that only binds DNA in the absence of ligand is converted to a ligand-dependent DNA binding protein.

Conjugados moleculares

Transfecc.

Liposomas catiónicos

Vector ideal

Capacidad de inserción

Irrestricto

Irrestricto

Irrestricto

1-1000 kb

Eficiencia de transferencia

Eficiente

Eficiente

Eficiente

Muy eficiente

Integración

No

No/Rara

No

Sí/No

Generación virus recombinantes

No

No

No

No

?

?

?

No

Expresión de proteínas virales

No

No

No

No

Expresión estable

Transitoria

Transitoria

Transitoria

Sí/No

?

Sí

Sí

Sí

No

Sí

Sí

Sí

VECTORES NO VIRALES

Oncogenicidad

Administración in vivo Transmisión a céls. quiescentes

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$ 320

TOTAL: $9,200

Just a few products could be produced by using both mammalian and microbial systems

The resulting proteins are found to have comparable structures and activity profiles although strictly speaking not being necessarily identical http://www.lonza.com/group/en/news/downloads/speeches.Par.0028.File2.tmp/part2.pdf

Mammalian system demands on the grow

http://www.lonza.com/group/en/news/downloads/speeches.Par.0028.File2.tmp/part2.pdf