pED vector, Transfection, Transduction,

1. MAMMALIAN CELL
EXPRESSION SYSTEM
Ramya R
Ph D I
Fish Biotechnology
IFPGS

2. Mammalian cell expression system (MES)
Special Features
Demand complex & specific media
Oxygen Supply
Low cell density
Slow growth Kinetics
High sensitivity to mechanical stress
Targets for adventitious virus
Require rigorous monitoring
Feature that makes MES more
preferable
• Protein production with accurate post-
translational modification ( glycosylation,
phosphorylation)
• Chaperone system
enables the above

3. Mammalian cell line
Characteristics
• Size - 10-50 µ
• Shape – Spherical
• Cell aggregation – single cell
• Doubling tine -24-48 hrs
• Shear sensitivity - Extremely high
• Oxygen uptake rate - 0.05-10 mmol/l/h
• Required KLα(Volumetric oxygen transfer
coefficient) in bioreactor operation
-0.25 – 10 h
• Protein localization - secreted
Widely used cell lines
Chinese hamster
ovary(CHO)
Mouse myeloma cells
(NS0 and Sp2/0 cells)
Human Embryonic
Kidney (HEK293)

4. Stable gene Expression(SEG)VsTransient gene Expression(TEG)
SEG: Integration of rDNA with the
genome of the cell line
Advantage:
All cell express the transgene
rDNA replicate along with genonomic
DNA, so no chance for lose
Yield as high as 5g/l in fed batch
culture.
TEG: Incorporation of rDNA into the
cytosol of cell line
Advantage:
There is no need to select the cells
with target rDNA.
The time frame fro transfection to
product purification is short
SEG TEG
Genetic Selection yes No
Time from DNA to product 6-12 months Upto 3 weeks
Specific Production
(pg/cell/day)
Up to 50 Below 10
Volumetric Productivity (g/l) Up to 5 0.02 to 0.08
Scalability Up to 20,000 l Up to 100 l
Application Large scale production of
therapeutic rec-protein
Small scale production of
protein for research

5. Mammalian ExpressionVector
Transcriptional Control element
a)Promoters and Enhancers
b)Introns
c)Polyadenylation signals
d)Transcription terminators
Polycistronic messages
a)Utilizing IRES elements
mRNA stability
a) Insertion of AU rich element
b) Substituting UTRs
c) Removal of destabilizing sequence
d) Using specific UTRs
e) Insertion of a minimal 8 residue
glycine –alanine repeat .
Fusion moieties
a) As Affinity handles
b) As reporter genes
c) As protein dimerization domains
Translational control elements
a) 5’ untranslated region
b)3’ untranslated region
c) Termination codon
c) Codon usage

6. Lentivirus Gene
ExpressionVector
Transcriptional Control element
RSV promoter
Promoter
mPGK Promoter, SV40 early pA
pUC ori
Translation control element
5' LTR-ΔU3
Kozak
3' LTR-ΔU3
mRNA Stability
WPRE
Polycistronic Messages
Marker
Ampicillin
Fusion Moieties
Ψ
RRE
cPPT

7. Key Component of pED
• AmpR- Ampicillin Resistant gene – selection
• SV40 – SimianVirus 40 promoter – Origin of
replication and enhancer
• AdMLP – Adenovirus major late promoter-
Transcription initiation site
• AdTPL- AdenovirusTripartite Leader –
Facilitate translation
• IVS- Intervening sequences or Introns –
mRNA stability
• SMC – Split marker mediated multiple piece
cloning- Assembles multiple DNA graments
into one construct
• EMC-L – Encephalomyocarditis Virus (EMC
& L- cell ribosomal entry site – help in DHFR
translation

8. Key Component of pED
Selection of
pED transfected
cell
•Utilize
fluorescent
derivative of
methotrexate
Stains DHFR
•Observe the
fluorescence
under
microscope
Cell sorting
• Use FACS to
isolate
transfected
cell
Selection of
pED transfected
cell
•Utilize
fluorescent
derivative of
methotrexate
Stains DHFR
•Observe the
fluorescence
under
microscope
Cell sorting
• Use FACS to
isolate
transfected
cell
• DHFR – Dihydrofolate reductase – selectable marker
• SV40 late pA – SimianVirus late polyadenylation
signal – Cleavage and polyadenylation signal
• VAI- AdenovirusVirus associated gene- to inihibit
protein Kinase
• pED is dicistronic –VAI gene and DHFR gene

9. Plasmid based
expression vector
Baculovirus
asVector
Vectors
Adenovirus
vectors
Vaccinia
vectors
Retroviral
Vector

10. Transfection: Process of introducing nucleic acid into eukaryotic cells using various
chemical or physical methods (non-viral method)
Factors AffectingTransfection
Cell Health
• Cells should be grown with
appropriate medium with all
necessary factors.
• Cells must be free of contamination.
• Fresh medium must be used if it
contains chemically unstable
components, such as thiamine.
• Cells should be incubated at 37°Cwith
CO2 supplied at the correct
percentage(5-10%) and 100%
relative immunity.
• Cells should be maintained in log
growth phase
Cell Culture
• Confluency and growth phase
• Cells should be transfected at 40-80% confluency
• Too few cells cause cell culture to grow poorly without cell to cell contact
• Too many cells result in contact inhibition, making cells resistant to uptake
of DNA and other macromolecules.
• Actively dividing cells take up DNA better than quiescent cell ( breakdown
and perforation of nuclear membrane during mitosis enable nuclear
delivery)
• Number of passages for primary cell line
• No. of passage < 50
• No. of passage of cell used in a variety of experiment should be consistent
• Cells may not response to the same transfection conditions after repeated
passage.

11. Factors Affecting transfection – DNA
quality and quantity
• Use high quality plasmid DNA that is free from protein, RNA and chemicals for
transfections; endotoxin removal must be a part of the procedure
• Typically, DNA is suspended in sterile water orTE buffer to a final concentration of
0.2 -1mg/ml.
• The optimal amount of DNA to use in transfection will vary widely depending up
on the type of DNA, transfection reagent/ method, target cell line and number of
cells.

12. TransfectionWorkflow
Add Nucleic acid
Analysis
Resuspend the cells in
electroporation buffer
Tissue culture - Count cells
Plate cells
Transfect the cells
Microscopy Flow cytometry
Reporter gene activity
Protein expression Gene Expression
Western blot Real time q PCR

13. Types ofTransfection
Reagent-Based Methods
• Lipids
• Calcium phosphate
• Cationic polymers
• DEAE- Dextrans
• Activated dendrimers
• Magnetic beads
Instrument based Method
• Biolistic technology
• Electroporation
• Microinjection
• Laserfection / optoinjection

14. Reagent Based method - Lipofection
Cationic Lipids: Liposomes /
Lipoplexes
■ Electrostatic interactions between
the positive charges of the cationic
lipid head groups and the negatively
charged phosphates of the DNA
backbone are the main forces that
allow DNA to spontaneously associate
with cationic lipids

15. Method
overview

16. Calcium Phosphate • The protocol involves mixing DNA
with calcium chloride, adding this in a
controlled manner to a buffered
saline/ phosphate solution, and
allowing the mixture to incubate at
room temperature.
• This step generates a precipitate that
is dispersed onto the cultured cells.
The precipitate is taken up by the
cells via endocytosis or phagocytosis.

17. Method Overview
• Solution A: DNA in calcium solution
• Solution B: 2x Hanks buffered saline solution
• Step 1: Add solution A to solution B while vortexing.
• Step 2:Incubate 20–30 min. Apply the solution to a
subconfluent cell culture;
• Step 3: Incubate 2–12 hr. Replace the solution with
complete growth medium
• Step 4: Assay for transient gene expression or begin
selection for stable transfection time.

18. Cationic Polymers
• Cationic polymers differ from cationic lipids in that they do
not contain a hydrophobic moiety and are completely
soluble in water. Given their polymeric nature, cationic
polymers can be synthesized in different lengths, with
different geometry (linear versus branched).
• Polyelectrolyte complex (PEC) formed between cationic
polymer and DNA tightly pack the DNA in PEC complex ,
thus shields from contact with Dnase.
• There are three general types of cationic polymers used in
transfections:
■■ Linear (histone, spermine, and polylysine)
■■ Branched
■■ Spherical
Cationic polymers include
polyethyleneimine (PEI) ,
Poly ( L-Lysine)
Cationic dendrimers.
Polybrene
Gelatin
Tetraminofullerene
Poly (L-histidine) graft poly (l-
Lysine)
Cationic polysaccharide

19. DEAE-dextran
• DEAE-dextran is a cationic polymer that
tightly associates with negatively charged
nucleic acids.
• The positively charged DNA:polymer
complex comes into close association with
the negatively charged cell membrane.
• DNA:polymer complex uptake into the cell
is presumed to occur via endocytosis or
macropinocytosis.

20. Method overview
• Solution A: DNA (~1–5 µg/ml) diluted into 2 ml of growth
medium with serum containing chloroquine
• Solution B: DEAE-dextran solution (~50–500 µg/ml)
• Solution C: ~5 ml of DMSO
• Solution D: Complete growth medium
• Step 1: Add solution A to solution B, then mix gently.
• Step2: Aspirate cell medium and apply the mixed A and B solutions
to the subconfluent cell culture. Incubate the DNA mixture for ~4
hr.
• Step3: Aspirate supernatant.
• Step4: Add solution C to induce DNA uptake.
• Step: Remove DMSO and replace with complete growth medium;
assay for transient gene expression.

21. Activated Dendrimers Structure
• Positively charged amino groups (termini) on the surface of
the dendrimer molecule interact with the negatively charged
phosphate groups of the DNA molecule to form a DNA-
dendrimer complex.
• The DNA-dendrimer complex has an overall positive net
charge and can bind to negatively charged surface molecules
on the membrane of eukaryotic cells.
• Complexes bound to the cell surface are taken into the cell by
nonspecific endocytosis.
• Once inside the cell, the complexes are transported to the
endosomes.
• Amino groups on the dendrimers that are unprotonated at
neutral pH can become protonated in the acidic environment
of the endosome.This leads to buffering of the endosome,
which inhibits pH-dependent endosomal nucleases.
•

22. Magnet-MediatedTransfection
• Magnet-mediated transfection uses magnetic force to deliver
DNA into target cells.
• Nucleic acids are first associated with magnetic nanoparticles.
Then, application of magnetic force drives the nucleic acid–
particle complexes toward and into the target cells, where the
cargo is released.
• Method Overview
• Dilute nucleic acid in medium.
• Add magnetic nanoparticle.
• Incubate 10–20 min.
• Add medium to adherent cells (2–4 x 105 cells).
• Add nucleic acid/nanoparticle solution.
• Place culture plate on magnet plate and Incubate 15 min.
• Remove magnet plate.

23. Electroporation
How ElectroporationWorks
• STEP 1: Electroporation exposes a cell to a high-
intensity electric field that temporarily destabilizes
the membrane.
• STEP 2: During this time the membrane is highly
permeable to exogenous molecules present in the
surrounding media.
• STEP 3: DNA then moves into the cell through these
holes.
• STEP4:When the field is turned off, the pores in the
membrane reseal, enclosing the DNA inside.

24. Type of Electrical Pulse — theWaveform
The most common electrical fields are exponential and square waveforms. The waveform has a significant effect on the transfection
efficiency for different cell types. Both exponential-decay and square-wave pulses have been used very effectively for electroporation.
Exponential Waveform
■■ Capacitors charged to a set voltage
■■ Set voltage is released from the selected
capacitor and decays rapidly (exponentially)
over time
Square Waveform
■■ Determined by pulse duration and/or
number of pulses
■■ Several short pulses may be more
beneficial than one long pulse

25. Biolistic Particle Delivery
Biolistic transformation is the delivery of nucleic acids into cells via high velocity nucleic
acid–coated microparticles.
Gene Gun
• Pressure range 100–600 psi enables fine-
tuning of penetration
• Highly portable — can be used in the field
• Small target area for accurate targeting
Biolistic Particle Delivery System
• Pressure range 450–2,200 psi gives flexibility
and penetration — ideal for plant applications
• Large target area — more cells can be
transformed

26. Gene Gun — Process Overview
• Step1: Precipitate DNA onto gold
particles.
• Step2: Load DNA/gold into tubing.
• Step3: Rotate tubing to coat DNA
gold over inside surface.
• Step4: Cut tubing into cartridges.
• Step5: Load cartridges into gene gun.
• Step 6: Deliver DNA into target cells.

27. BiolisticTechnology- Helium powdered acceleration system
• DNA-coated gold particles (microcarrier)
are spread over the central area of a thin
plastic disk (macrocarrier).
• Disk loaded with the DNA-gold particles is
placed into a holder inside the He system.
• The system uses high-pressure helium,
released by a rupture disk, and partial
vacuum to propel the macrocarrier loaded
with microcarrier toward the target cells.
• Macrocarrier is stopped after a short
distance by a stopping screen.
• DNA-coated gold particles continue
travelling toward the target to penetrate
the cells.
• Sample chamber is subjected to partial
vacuum, from 15 to 29 inches of mercury,
depending on the target cells.

28. Microinjection
■■ Direct injection of naked DNA
■■ Laborious (one cell at a time)
■■ Technically demanding and costly
■■ Can be used for many animals
Specific application like ectopic gene
transfer, microinjection is potentially
viable.

29. Laserfection / Optoinjection
• This procedure uses laser light to transiently
permeabilize a large number of cells in a very short
time
• Various substances can be efficiently optoinjected,
including ions, small molecules, dextrans, short
interfering RNAs (siRNAs), plasmids, proteins, and
semiconductor nanocrystals, into numerous cell
types.
■■ Advantages: very efficient; works
with many cell types; fewer cell
manipulations needed
■■ Disadvantages: requires the cells
to be attached; expensive laser-based
equipment needed

30. Transduction: the process by which foreign DNA is introduced into a cell
by a virus or viral vector.
• A plasmid is constructed in
which the genes to be
transferred are flanked by
viral sequences that are
used by viral proteins to
recognize and package the
viral genome into viral
particles.

31. Viral workflow

32. ViralVectors
Retroviruses
■■ Murine leukemia virus (MuLV)
■■ Human immunodeficiency virus
(HIV)
■■ HumanT-cell lymphotropic virus
(HTLV)
DNAViruses
■■ Adenovirus
■■ Adeno-associated virus (AAV)
■■ Herpes simplex virus (HSV)
• Retroviruses — a class of viruses that can create double-
stranded DNA copies of their RNA genomes; these
copies can be integrated into the chromosomes of host
cells. HIV is a retrovirus.
• Adenoviruses — a class of viruses with double-stranded
DNA genomes that cause respiratory, intestinal, and eye
infections in humans.The virus that causes the common
cold is an adenovirus.
• Adeno-associated viruses — a class of small, single-
stranded DNA viruses that can insert their genetic
material at a specific site on chromosome 19.
• Herpes simplex viruses — a class of double-stranded
DNA viruses that infect a particular cell type, neurons.
Herpes simplex virus type 1 is a common human
pathogen that causes cold sores.

33. Viral Attributes

34. Application of Mammalian Cell Expression system
• Production of monoclonal antibody
• Production of Urokinase
• Production of follicle stimulating hormone
• Production of viral vaccine
• Production of various therapeutic proteins

35. DISCUSSION