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Molecular methods of cell culture II
Expression of mammalian expression vector in cell culture
-Galactosidase Reporter system
Hydrolaze enzyme catalyze hydrolysis of -galactoside to
monosaccharide
 Contain 1023 a.a.
Mammalian -Galactosidase Reporter system
Expression of -Galactosidase in WI-380 cells
Tet on Inducible system
Induction by Doxicyclin
TRE: Tetracyclin( Doxicyclin) response element
rtTA : regulatory protein
ubiquitous promoter (PU) or tissue- or cell type-specific promoter (PTS)
Gene of interest is under the regulation of the rtTA-dependent promoter
Acta Biochimica et Biophysica Sinica 2007, 39(4): 235–246
Expression of Fluorescent protein in cell culture
Green fluorescent protein ( GFP) 238 a.a or Red Fluorescent
protein
Cloned from jelly fish( Aequorea victoria)
Red Fluorescent protein
fusion protein to the
multiple cloning site
GFP
DAPI
HEL: Human Embryonic Lung
RFP
DAPI
HEL: Human Embryonic Lung
Promoter reporter system
Luciferase assay
MCS
SV40
Luciferase
Multiple cloning site
Luciferase gene
NON-VIRAL GENE DELIVERY
1. Synthetic Polymers
To generate cationic polymers to interact electrostatically with
and neutralize negatively charged DNA
 Poly L-lysine (PLL)
high cytotoxicity
aggregate and precipitate
polyethylene glycol (PEG)
flexible, water-soluble
----Polymer covalent coupling of PEG( PEGylation) of a target
molecule
i.e. PLL : cytotoxicity and non-specific protein adsorption
i.e. polyethyleneimine (PEI), a cationic gene carrier with superior
transfection efficiency and unique buffering properties
2. Natural Polymers
cyclodextrin, chitosan, collagen, gelatin, and alginate
Advantage:
Innate environmental responsiveness
Ability to be degraded
Remodeled by cell-secreted enzymes.
Non-toxic at both low and high concentrations, are
Readily incorporated into oral or bolus matrix delivery
systems,
Serve as tissue engineering scaffolds
Entry of DNA-Polymer Complex
DNA :polymer
complex
DNA :polymer
complex
DNA :polymer
complex
endosome
Episomal or
integration with selection
Transcription
Virus vector
 Gene delivery in cell culture system
 Deliver vector for gene therapy
 Recombinant protein production in industry
Clinical application of viral vector
Adenovirus(
Tumors,
Haemopoietic
cells
AAV( liver,
muscle,
retinal
Polymer
coated
adenovirus
( tumors)
Retrovirus( t
umor,stem,h
aemopoitic
cells
Herpes
simplex
virus(CNS,
PNS,muscle,
Haemopoiet
ic, stem
cells
Alpha
virus
( tumors)
Lentivirus(
CNS,
liver,muscl
e
Liposome
encapsula
te alpha
virus
TRENDS in Biotechnology Vol.21 No.3: 119 2003
Requirements for viral gene therapy vectors
1. Delivery system must be safe and immunologically inert.
2. Protect the genetic material from degradation.
3. Vector must encode an effective therapeutic gene that has
sustained expression at a defined target site
4. Tissue-specific targeting
5. Site-specific chromosomal integration
6. Controlled infection of both dividing and non-dividing cells
Virus Vectors
retrovirus
Recombinant cell lines
SV-40
 substitute virus gene with foreign genes
( supply virus missing gene by cotransfection with
helper virus)
 Infect monkey cells only
 Carry smaller size of foreign genes
SV-40
T ag integration
T ag protein allows
replication of plasmid
Vaccinia virus
 Carry smaller size of foreign genes
 DNA recombination occurs in the cells
 Virus replicate within the cytoplasm of the host cells
 Higher level of protein expression
Baculovirus
 Foreign gene maybe coexpressed with
structural gene ( structural protein expresses when
infection occurs)
replacement of coat protein gene
with gene of interest
Baculovirus
Replace coat protein gene
with gene of interest
Developing baculovirus-insect cell expression system for
humanized recombinant glycoprotein
Baculovirus-insect cell expression system
4. Retrovirus
Recombinant retroviruses used in gene delivery:
1.γ-retroviruses
2. Lentiviruses
3. spumaviruses
Retrovirus life cycle
Retroviral vector development for increased efficiency and targeting
Structure of a simple retroviral
genome containing coding sequences for gag, pro, pol, and env for
replication
att
att
U3
R
U5 PBS

gag
pro
pol
env
5’LTR
PPT U3
R
U5
3’LTR
Genome 7-10kb
 contain gag, pro, pol, env :
encode structural capsid proteins, viral protease,
integrase, and viral reverse transcriptase, enveloped glycoproteins
Retroviral vector development for increased efficiency and targeting
att
U3
att
att
R U5 PBS

gag
pro
pol
env
PPT U3
5’LTR
U3
R
5’LTR
U5 PBS

R U5
att
3’LTR
cPPT
Transgene
U3
R
U5
P
ampr
3’LTR
 Replication coding sequences removed and transgene inserted.
 U3 component of the 5′LTR is used as a promoter to drive transgene
expression
 P: Heterologous or tissue-specific promoter inserted to drive an
ampicillin gene to facilitate ex vivo selection of transduced cells.
LTRs: dual long-terminal repeats , (PBS): primer binding site ,
Ψ: signal, (att): attachment sites polypurine tract : (PPT).
Retroviral vector development for increased efficiency and targeting
Structure of an enhanced self-inactivating retroviral gene therapy vector
att
U3
att
att
R U5 PBS

gag
pro
pol
env
PPT U3
5’LTR
RSV R U5 PBS
5’LTR

R U5
att
3’LTR
cPPT
P Transgene
CI,CR,WPREs/MAR,ampr
 substitution of the 5′LTR U3 component by RSV.
U3
R U5
3’LTR
 P: internal promoter is tissue-specific to limit transgene expression.
 Additional genetic elements—incorporated to enhance site specificity and
integration efficiency while limiting
CI : chromatin insulators , limit position effect variegation
CR: chromatin structure regulators
WPRE : woodchuck hepatitis virus post-transcriptional regulatory element,
enhance mRNA transcript stability
S/MAR : scaffold or matrix attachment regions or resistance, anchorage of
chromatin with stabilization of chromosomal loops
Function of 3rd generation MLV packing cell line
Therapeutic gene
Stable integration
Advantages of retrovirus vector
Stably traduce dividing cells
 Long term transgene expression
 RNA virus ( virus genome may be integrated into the host genome)
 Infect various kinds of mammalian cell lines
 Infection of mammalian cells by retrovirus does not cause host
death
 Carry -galactosidase gene
 Viral gene expression is driven by stronger promoter
 Low immune response
Disadvantage of retrovirus vector
 Low viral titer
 Only infecting dividing cells
 Integrative may activate or damage cellular genes
 Random insertion into host cell and causes
 oncogenic activation or tumor-suppressor gene
 Limited insert capacity( 8kb)
 Inactivation by human complement
 Inability to transduce nondividing cells
Lentivirus
human immunodeficiency virus (HIV)
simian immunodeficiency( SIV)
non-primate- equine infectious anaemia virus (EIAV)
feline immunodeficiency virus (FIV)
Recombinant lentiviral (rLV) vector systems
tool to achieve high transduction efficiency for non-dividing cells
e.g. central nervous system, retinal cells, pancreatic islets, progenitor and
differentiated hematopoietic cells
Accessory and Regulatory genes
 oncogenesis (tat), apoptosis (vpr), MHC downregulation, (nef )
differentiation (vpu)
 Biologically active and can cause detrimental effects on cells
Lenti virus vectors lacking tat, vpr, nef vpu
TU, transducing units; p, promoter; env, envelope; WPRE, Woodchuck hepatitis posttranscriptional regulatory element; cPPT, central polypurine tract; CMVp, cytomegalovirus
promoter
Lentiviral transduction of quiescent T-cells
The T-cell-specific stimulation then allows reverse-transcription of the
viral genome followed by nuclear import and integration of the proviral
DNA
J Gene Med 2004; 6: S83–S94.
Retroviral Transduction of T-cell Receptors in Mouse T-cells
http://www.jove.com/details.php?id=2307
Adeno virus
Largest non-enveloped virus and contain linear, double-stranded
Genome :36kB
ITR
Early
E1A
Late
ITR
Early genes : function as regulatory proteins for viral replication
Late genes : encode structural proteins for new virus assembly
E1A gene: a trans-acting transcriptional regulatory factor that is required
for early gene activation
CAR receptor
pH dependent
release of virus
particle
Adenovirus Serotype
Group C: most common in nature and in adenoviral gene therapy are human
serotypes 2 and 5
Genome type of Adenovirus and different types of Ad5 derived vector
E1A E1B
E3
MLP
L1
L2
L3
L4
L5
Ad5 genome
E2B
E2A
E4
Transgene
1st generation
E1(+/-)-deleted
Immunogenic
response
Transgene
2nd generation
E1(+/-E3)+E2/4-deleted
E1A
Transgene I
Transgene II
High-capacity vector
E1A
Non-coding stuffer DNA
gutless
Recombinant Adenovirus propagated in the cell line expressing E1
region
Advantage of Adenovirus:
Relatively simple genetics
 Can accommodate large insert allows controllable expression
 High viral titer
 Infect non-dividing cell
Disadvantage of Adenovirus :
 Non-integrative
 Transient expression
 Immunogenic response observed
Adeno Associated vector
ITR 145 bp
rep
cap
ITR 145 bp
r AAV production
Transcription unit
ITR
ITR
rep
cap
Helper adenovirus
293
cell
Mixed helper /r AAv
Heat 56oC
CsCl2 gradient centrifugation
Recombinant AAV
Advantage of Adeno associated virus:
Parvoviridae family
Non human disease associate
 Integrate stably into chromosome 19( site specific
integration)
Transduce mitotic and post mitotic
Disadvantage of Adeno associated virus:
 Limited host range
High-Efficiency Transduction of Liver Cancer Cells by Recombinant
Adeno-Associated Virus Serotype 3 Vectors
https://www.jove.com/details.php?id=2538
Gene therapy for bone regeneration
Scheller & Krebsbach etal , J Dent Res 88(7) 2009