Transferencia de Genes a Células Animales en Cultivo 2011-3.pdf · Transferencia de Genes a...

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 aCCéélulas Animales en Cultivolulas Animales en Cultivo

Conceptos y TConceptos y Téécnicas de Biotecnologcnicas de Biotecnologíía I a I 2011 2011 –– 2do cuatrimestre2do cuatrimestre

FBMCFBMC--FCENFCEN--UBAUBA

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•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 ProductionIn 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



• Initial groups can be trimmed and then expanded to create different final modifications



• 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 inCulture

• 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 andmammalian 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 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


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) )



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 + h (luz)

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

XFP + h1 (luz) XFP + h2 (luz) 1 >2 Selector: FACS


Fluorescence-activated cell sorter


Reporter gene systems1. 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 camerasallow 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 harmlessfor 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



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)DHFTHF½ 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 liposomepreparation


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 productsto 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 expressionof 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 activityconstitutivepromoters

a ubiquitous low level promoter that is used to construct reporter genes

deleted Drosophila alcohol dehydrogenase promoter

Minimal promoter


Generalized Eukaryotic CloningVector

• Prokaryotic origin of replication, selectable marker

• Eukaryotic origin, selectable marker

• MCS with eukaryotic promoter and transcriptional terminator/polyadenylation 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 repressedin 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 partTET-OFF system


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

the 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


Top Fifteen Biopharmaceuticals (1999 Sales)

Epogen Amgen Erythropoeitin $1,770Neupogen Amgen G-CSF $1,245Infergen Schering-Plough Interferon $1,010 Humulin Eli Lilly Insulin $ 880Avonex Biogen Interferon 1a $ 570Engerix SmithKline Beecham Hepatitis B vaccine $ 505KoGENate Bayer Corporation Factor VIII $ 470Cerezyme Genzyme Tissue Repair Glucocerebrosidase $ 430Betaseron Berlex Laboratories Interferon 1b $ 395GenoTropin Pharmacia & Upjohn Human growth hormone $ 380Enbrel Immunex TNF receptor $ 340ReoPro Centocor Monoclonal antibody $ 335Gonal-F Serono Laboratories Follicle stimulating hormone $ 320RECOMBIVAX HB Merck Heptatis B vaccine $ 275Synagis MedImmune Monoclonal antibody $ 275

TOTAL: $9,200


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

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 thegrow

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