Download Transferencia de Genes a Células Animales en Cultivo II

Conceptos y Técnicas de Biotecnología I
2016 – 2do cuatrimestre
FBMC-FCEN-UBA
Transferencia de Genes a
Células Animales en Cultivo II
Unidad de Transferencia Genética
Instituto de Oncología “Ángel H. Roffo”
Universidad de Buenos Aires
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
No
No
No
No
Transitoria
Transitoria
Transitoria
Sí/No
?
Sí
Sí
Sí
No
Sí
Sí
Sí
VECTORES
NO VIRALES
Oncogenicidad
Expresión de
proteínas virales
Expresión estable
Administración
in vivo
Transmisión a
céls. quiescentes
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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