Unidad de Transferencia GenUnidad de Transferencia GenééticaticaInstituto de OncologInstituto de Oncologíía a “Á“Ángel H. ngel H. RoffoRoffo””

Universidad de Buenos AiresUniversidad de Buenos Aires

Transferencia de Genes aTransferencia de Genes a

CCéélulas Animales en Cultivolulas Animales en Cultivo

Conceptos y TConceptos y Téécnicas de Biotecnologcnicas de Biotecnologíía I a I 2012 2012 –– 2do cuatrimestre2do cuatrimestre

FBMCFBMC--FCENFCEN--UBAUBA

•BACTERIAS (i.e.: Escherichia coli)•HONGOS (i.e.: Saccharomyces cereviseae)•CULTIVOS CELULARES (animales o vegetales)•PLANTASALGASVASCULARES•ANIMALES:PECESAVESMAMÍFEROSBovinosCaprinosOvinosPorcinosHUMANOS: Transgénesis parcial � Terapia Génica

ORGANISMOS TRANSGÉNICOS- INVOLUCRADOS EN BIOTECNOLOGÍA -

Transferencia de Genes a CTransferencia de Genes a Céélulas Animales en Cultivolulas Animales en Cultivo

PROPÓSITOS

•Confirmar identidad de genes

•Caracterizar oncogenes

•Expresar proteínas que necesitan modificaciones post-

traduccionales

•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

Chemically defined media (Eagle, Earle)ConsistencySterilizationReduced chance of contamination

50-s

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.

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

0.0940.094L-Valine

0.63510.10379L-Tyrosine • 2Na •2H2O

0.0160.016L-Tryptophan

0.0950.095L-Threonine

0.0420.042L-Serine

0.0660.066L-Phenylalanine

0.03—L-Methionine

0.1461.46L-Lysine • HCl

0.1050.105L-Leucine

0.1050.105L-Isoleucine

0.0420.042L-Histidine • HCl • H2O

0.030.03Glycine

0.0626—L-Cystine • 2HCl

0.0840.084L-Arginine • HCl

Amino Acids

0.1090.109Sodium Phosphate Monobasic (anhydrous)

6.46.4Sodium Chloride

3.73.7Sodium Bicarbonate

0.40.4Potassium Chloride

0.097670.09767Magnesium Sulfate (anhydrous)

0.00010.0001Ferric Nitrate • 9H2O

0.20.2Calcium Chloride

Inorganic Salts

Ejemplo de medio de cultivo de células de mamífero:

Dulbecco Modified Eagle medium (DMEM)

0.5840.584L-Glutamine

Add

—0.11Pyruvic Acid • Na

—0.0159Phenol Red • Na

4.54.5D-Glucose

Other

0.0040.004Thiamine • HCl

0.00040.0004Riboflavin

0.0040.004Pyridoxine • HCl

——Pyridoxal • HCl

0.0040.004D-Pantothenic Acid(hemicalcium)

0.0040.004Niacinamide

0.00720.0072myo-Inositol

0.0040.004Folic Acid

0.0040.004Choline Chloride

Vitamins

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.

0.0

0.0

0.0

0.0

0.1

1.0

10.0

0 50 100Time (h)

Cx

(g/l

)

YeastHybridoma

Growth curves in yeasts and

mammalian cells are different

Minimumdensity Lag phase

exponential

Stationary

decline

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 microcarriers

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

Canine KidneyEpithelial

Common cell lines

CHO

HeLa

MDCK

BHK

Vero

WI-38

3T3

Chinese Hamster OvaryEpithelial

Mouse fibroblastFibroblast

Monkey KidneyFibroblast

Epithelial Human cervical carcinoma

Human fetal lungFibroblast

Baby Hamster KidneyFibroblast

MARCADORES FENOTMARCADORES FENOTÍÍPICOSPICOS

••Crecimiento en agarCrecimiento en agar••Crecimiento con bajo sueroCrecimiento con bajo suero••Cambios morfolCambios morfolóógicosgicos••Rescate de la muerte de cultivos celulares primariosRescate de la muerte de cultivos celulares primarios

MARCADORES BIOQUMARCADORES BIOQUÍÍMICOSMICOS

IndicadoresIndicadores••Anticuerpos especAnticuerpos especííficosficos••ChloramphenicolChloramphenicol acetyltransferaseacetyltransferase ((catcat))••ββ--galactosidasegalactosidase ((ββ--galgal))••LuciferaseLuciferase

SelectoresSelectores••ThymidineThymidine kinasekinase ((tktk))••XantineXantine guanineguanine phosphoribosylphosphoribosyl transferasetransferase ((xgprtxgprt))••AminoglicosideAminoglicoside phosphotransferasephosphotransferase ((aphtapht//neoneorr))••DihydrofolateDihydrofolate reductasereductase ((dhfrdhfr))••HygromycinHygromycin B B phosphotransferasephosphotransferase ((hyghygrr)

Indicadores/SelectoresIndicadores/Selectores••GreenGreen/Blue/Red /Blue/Red fluorescencefluorescence proteinsproteins ((gfpgfp//bfpbfp//rfprfp) )

MARCADORES BIOQUMARCADORES BIOQUÍÍMICOSMICOS

IndicadoresIndicadores

•Anticuerpos específicosAg + AbI � Ag.AbIAg.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 + glucosaONPG � o-nitrofenol (amarillo) + galactosaXgal � X (azul) + galactosa

•LuciferaseLuciferina + ATP � Luciferina + ADP + hν (luz)

Indicadores/SelectoresIndicadores/Selectores•Green/Blue/Red fluorescence proteins (gfp/bfp/rfp)

XFP + hν1 (luz) � XFP + hν2 (luz) νννν1 >νννν2 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)

For mammalian cells

it is laborous and expensive.

Extract protein and measure activity…

thin-layer chromatographic sheetChloramphenicol

is radiolabelled

β-galactosidase (βgal) systems

luciferase (luc) systemsfirefly species Photinus pyralis

oxidation of compounds called luciferans( ATP-dependent process)

luciferans emit fluorescense

Expressed luciferase catalyses

mouse with a strain of salmonella

Mice are injected

with LUC+ salmonellas.

Sensitive digital cameras

allow non-invasive detection.

For GT vectors pictures

look the same

luminometer measurement

Green fluorescent protein (GFP)autofluorescent protein from Pacific Northwest jellyfish Aequorea victoriaGFP is an extremely stable proteinof 238 amino acids with unique post-translationallycreated 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 BIOQUMARCADORES BIOQUÍÍMICOSMICOS

SelectoresSelectores

•Thymidine kinase (tk):)dT +ATP ����dTMP +ADP ½ select. céls. tk-: HAT (hipoxantina, aminopterina, timidina)dUMP ���� dTTP inhib: aminopterina

•Xantine-guanine phosphorybosil transferase (xgprt)Xantina →→→→XMP →→→→ GMP ←←←← ←←←← guanina ½ selectivo p/toda cél.: AAMX

↑↑↑↑ inhib: ac. aminofenólico (adenosina, aminopterina,precursores →→→→ →→→→ IMP inhib: aminpoterina ac. Aminofenólico, xantina)

↓↓↓↓ASMP →→→→ AMP ASMP: ac. adenilosuccínico

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

•Dihydrofolate reductase (dhfr)DHF→→→→THF½ selectivo en céls. dhfr-: ausencia de nucleósidos½ selectivo p/toda cél.: methotrexate (MTX)

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

Gene amplification

• Vectores virales

• Vectores no virales

TRANSFERENCIA GENTRANSFERENCIA GENÉÉTICA:TICA:METODOLOGIAMETODOLOGIA

Direccionamiento de vectores

• Via de administración

• Receptores celulares

• Promotores específicos de tejido

•• Vectores ViralesVectores 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 cDNAexpression cassette (adenovirusITR, E1 enhancer, adenovirus encapsidation signal, CMV promoter, and SV40 a polyadenylation signal). Homologous recombination sequences arealso 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 approximatemap units. This recombinant genome is replication defective. However, in the presence of E1 proteins (provided in trans by 293 cells), the recombinantgenome 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

VECTORES VIRALES

Retrovirus Adenovirus Herpes virus Virus Adenoasociados

Tamaño máximo del gen terapeútico

8 kb 35 kb 30 kb 4,8 kb

Células objetivo Sólo en división activa

En división activa o sin

división

En división activa o sin

división

En división o quizás sin división

Administración Ex vivo o in

situ

Ex vivo o in

situ

Ex vivo o in

situ

Ex vivo o in

situ

Expresión del gen terapeútico

Estable Transitoria Transitoria Posiblemente estable

Indice de expresión del gen terapeútico

Moderado Elevado Moderado Moderado

Riesgos Posible integración mutagénica

Reacciones inflamatorias/inmunitarias

Posible integración mutagénica

Posible integración mutagénica

Inmunidad preexistente en el hospedador

No Sí Sí Sí

Recombinación con el hospedador

Improbable Posible Posible Improbable

Recombinación con el virus parental

Imposible Posible Posible Posible

•• VectoresVectores no no ViralesVirales

•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

TransfectionMicroinjection

Lipofection Electroporation

Liposomes

Why naked DNA?

Lets’ wrap it in something safe

to increase transfection rate

Therapeutic drugs

Lipids – is an obvious idea !

Liposomes are formed by the self-assembly of phospholipid

molecules in an aqueous environment.

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

Anionic liposome

Cationic liposomes

positively charged lipid dropletscan interact with negatively charged DNA

to wrap it up and deliver to cells

Positively charged lipid heads

Lipofectin, lipofectamine, lipofectase….

Inside liposomes DNA is resistant to degradation

Lab procedure for liposome

preparation

Lípidos catiónicos: Citofectinas

Complejo DNA/lípido catiónico: Lipoplex

Electron photomicrographs of lipid-DNA complexes.

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

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

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 transfectedwith 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 / Lipids / PolycationsPolycations / DNA (LPD) Complexes/ 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

Moderately high level gene expression in

mammalian cells

simian virus 40 early

enhancer/prom. SV40

Regulated by doxycycline (tetracycline)TET-off and TET-on systems

based on tet-resistance

operon of the E. coli Tn10

transposon

steroid-regulated gene expression in mammalian cells

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

Regulated

promoters

targeted expression of genes to mouse thymocytes for immunological studies

lymphocyte-specific tyrosine kinasepromoter (LCK)

targeted expression of genes to mammary

tissue in animals

whey acidic protein promoter (WAP)

Cell-specific

promoters

high level gene expression in mammalian

cells

cytomegalovirus

immediate early promoter

(CMV)

High activity

constitutive

promoters

a ubiquitous low level promoter that is used to construct

reporter genes

deleted Drosophila alcohol

dehydrogenase promoterMinimal promoter

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 systemsManfred Gossen and Hermann Bujard

www.biochem.arizona.edu

control the expression of genes that have been cloned downstream of a

promoter containing tetracycline receptor (TetR) binding sites.

The "Tet-off" system is repressed

in the presence of the doxycycline

VP is derived from the herpes simplex virus VP16 protein.

TET-VP producing vector

Gene of interest

expressing vector

VP – RNA pol interacting part

TetR - tet binding part

TET-OFF system

"Tet-on" system is activated in the presence of

doxycyclinethe DNA binding domain of the Tet-on regulator (rTetR) contains mutations

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

RNA-pol

VECTORESNO VIRALES

Conjugadosmoleculares

Transfecc. Liposomascatiónicos

Vector ideal

Capacidad de inserción

Irrestricto Irrestricto Irrestricto 1-1000 kb

Eficiencia detransferencia

Eficiente Eficiente Eficiente Muy eficiente

Integración No No/Rara No Sí/No

Generación virus recombinantes

No No No No

Oncogenicidad ? ? ? No

Expresión deproteínas virales

No No No No

Expresión estable Transitoria Transitoria Transitoria Sí/No

Administraciónin vivo

? Sí Sí Sí

Transmisión acéls. quiescentes

No Sí Sí Sí

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The resulting proteins are found to have comparable structures and activity profiles

although strictly speakingnot being necessarily identical

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

Mammalian system demands on the

grow

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