Download MRT lec 6

Course Contents
1.
2.
3.
4.
5.
6.
7.
8.
DNA and RNA isolation
Quantification of DNA and RNA
Primer designing
PCR
Electrophoresis
Sequencing
Karyotyping
Restriction Mapping
9. Flow cytometry
10.Hybridization
a. Western blotting
b. Southern blotting
c. Northern blotting
d. FISH
11.Transfection
21.Tissue culturing
12.Transduction
22.Slide Preparation and Cell Stains
13.Transformation
23.Agar plate preparation and streaking for
14.Cloning
the purpose of individual colony isolation
15.Microarrays
24.Bacterial Growth on selective agar
16.Chromatography
25.Quantification: Colony Forming Units
17.Immunochemistry
(CFU)
18.ELISA
26.Dilution Plating
19.Bioinformatics and techniques
27.Identification
20.Ethical issues
colonies
and
characteristics
of
Assignment
Presentation
Quiz
Transfection :
is the process of deliberately introducing nucleic acids into cells.
Transformation:
non-viral DNA transfer in bacteria, non-animal eukaryotic cells and plant cells.
Transduction: is often used to describe virus-mediated DNA transfer.
What to transfer
Genetic material (such as supercoiled plasmid DNA or siRNA constructs), or
even proteins such as antibodies, may be transfected.
Target:
Transfection of animal cells typically involves opening transient pores or "holes" in
the cell membrane, to allow the uptake of material.
TRANSFECTION
Transient Transfection
Rapid, scalable, high-yield protein production from transiently transfected suspension
cultures.
the DNA introduced in the transfection process is usually not integrated into the
nuclear genome, the foreign DNA will be diluted through mitosis or degraded.
Stable Transfection
Stable transfection introduces DNA into cells long-term and pass the introduced DNA
to their progeny.
marker gene is co-transfected, which gives the cell some selectable advantage, such as
resistance towards a certain toxin
Common Marker Genes
Gene name
Gene product
Assay
LacZ
β-galactosidase
enzyme assay or
Histochemical
cat
Chloramphenicol
acetyltransferase
Chloramphenicol resist
ant
neo
Neomycin phosphotra
G418 resistant
nsferase
gfp
Green fluorescent
protein
Fluorescent
rfp
Red fluorescent
protein
microscopical, spectro
photometry
CHEMICAL-BASED TRANSFECTION
calcium phosphate, HEPES((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid )-buffered
saline solution (HeBS) containing phosphate ions is combined with a calcium chloride solution
containing the DNA to be transfected. When the two are combined, a fine precipitate of the
positively charged calcium and the negatively charged phosphate will form, binding the DNA
to be transfected on its surface. The suspension of the precipitate is then added to the cells to be
transfected (usually a cell culture grown in a monolayer). By a process not entirely understood,
the cells take up some of the precipitate, and with it, the DNA.
• highly branched organic compounds, so-called dendrimers, to bind
the DNA and get it into the cell.
•Liposomes: small, membrane-bounded bodies that are in some ways similar to the
structure of a cell and can actually fuse with the cell membrane, releasing the DNA into
the cell.
Liposomes can be created by sonicating phosphatidylcholine rich phospholipids in water.
Low shear rates create multilamellar liposomes, which have many layers like an onion.
Continued high-shear sonication tends to form smaller unilamellar liposomes. In this
technique, the liposome contents are the same as the contents of the aqueous phase.
Sonication is generally considered a "gross" method of preparation as it can damage the
structure of the drug to be encapsulated.
NON-CHEMICAL TRANSFECTION
•Electroporation transient increase in the permeability of cell membrane is achieved
when the cells are exposed to short pulses of an intense electric field.
Sonoporation uses high-intensity ultrasound to induce pore formation in cell membranes.
•Optical transfection is a method where a tiny (~1 µm diameter) hole is transiently generated
in the plasma membrane of a cell using a highly focused laser.
•Protoplast fusion is a technique in which transformed bacterial cells are treated with
lysozyme in order to remove the cell wall. Following this, fusogenic agents (e.g., Sendai
virus, PEG, or electroporation) are used in order to fuse the protoplast carrying the gene of
interest with the target recipient cell. A major disadvantage of this method is that bacterial
components are non-specifically introduced into the target cell as well.
•Impalefection is a method of introducing DNA bound to a surface of a nanofiber that is
inserted into a cell. This approach can also be implemented with arrays of nanofibers that
are introduced into large numbers of cells and intact tissue.
PARTICLE BASED TRANSFECTION
• GENE GUN: where the DNA is coupled to a nanoparticle of an inert solid
(commonly gold) which is then "shot" directly into the target cell's nucleus.
Magnetofection, Nucleic acids are first associated with magnetic nanoparticles.
Then, application of magnetic force drives the nucleic acid particle complexes towards
and into the target cells, where the cargo is released.
Impalefection is carried out by impaling cells by elongated nanostructures and
arrays of such nanostructures such as carbon nanofibers or silicon nanowires which
have been functionalized with plasmid DNA.
•particle bombardment. The nucleic acid is delivered through membrane penetration at
a high velocity, usually connected to micro projectiles.
TRANSDUCTION
Introduced of DNA into cells using viruses as a carrier
Viral vectors are tailored to their specific applications but generally share a few key
properties.
Safety:
modified in such a way as to minimize the risk of handling them by deletion of a part of the
viral genome critical for viral replication.
Low toxicity: The viral vector should have a minimal effect on the physiology of the cell it
infects.
Stability:
Some viruses are genetically unstable and can rapidly rearrange their
genomes. This is detrimental to predictability and reproducibility of the work conducted using
a viral vector and is avoided in their design.
Cell type specificity: Most viral vectors are engineered to infect as wide a range of cell
types as possible. However, sometimes the opposite is preferred. The viral receptor can be
modified to target the virus to a specific kind of cell. Viruses modified in this manner are said
to be pseudo typed.
Identification:
Viral vectors are often given certain genes that help identify which cells
took up the viral genes.
APPLICATIONS
Basic research
Gene therapy
vaccines
Immortalization of primary T cells
LNGFR
hTERT
VIRAL VECTOR xlox(NGFR)TERT
GP2-293
GP2xTERT11 PRODUCER CELL LINE
ENVELOPE CONSTRUCT
PACKAGED TERT
RECOMBINANT VECTOR
Staining with anti-NGFR ab beads
N
S
PRIMARY T CELLS
Retroviruses
 number of FDA-approved clinical trials such as the SCID-X1 trial.
 either be replication-competent or replication-defective..
involves the requirement for cells to be actively dividing for transduction.
cells such as neurons are very resistant to infection and transduction by
retroviruses.
There is concern that insertional mutagenesis due to integration into the
host genome might lead to cancer or leukemia
LENTIVIRUSES
Lentiviruses are a subclass of Retroviruses.
able to integrate into the genome of non-dividing cells, The site of integration is
unpredictable,
studies have shown that lentivirus vectors have a lower tendency to integrate in
places that potentially cause cancer , clinical trials that utilized lentiviral vectors to
deliver gene therapy for the treatment of HIV experienced no increase in mutagenic
or oncologic events.
One or more plasmids, generally referred to as packaging plasmids, encode
the virion proteins, such as the capsid and the reverse transcriptase. Another plasmid
contains the genetic material to be delivered by the vector. It is transcribed to produce
the single-stranded RNA viral genome and is marked by the presence of the ψ (psi)
sequence. This sequence is used to package the genome into the virion.
ADENOVIRUSES
As opposed to Lentiviruses, adenoviral DNA does not integrate into the
genome and is not replicated during cell division.
with adenoviruses, which cause respiratory, gastrointestinal and eye infections,
they trigger a rapid immune response with potentially dangerous consequences.
To overcome this problem scientists are currently investigating adenovirusesto
which humans do not have immunity.
ADENO-ASSOCIATED VIRUSES
AAV is not currently known to cause disease and consequently the virus
causes a very mild immune response. AAV can infect both dividing and
non-dividing cells and may incorporate its genome into that of the host
cell. These features make AAV a very attractive candidate for creating viral
vectors for gene therapy