Properties of Adult SC

A stem cell possesses two properties:

Self-renewal, which is the ability to go through numerous cycles of cell division while
still maintaining its undifferentiated state.
multipotency or multidifferentiative potential, which is the ability to generate
progeny of several distinct cell types, (for example glial cells and neurons) as opposed
to unipotency, which is the term for cells that are restricted to producing a single-cell
type. However, some researchers do not consider multipotency to be essential, and
believe that unipotent self-renewing stem cells can exist.
These properties can be illustrated with relative ease in vitro, using methods such as
clonogenic assays, where the progeny of a single cell is characterized. However, it is
known that in vitro cell culture conditions can alter the behavior of cells. Proving that
a particular subpopulation of cells possesses stem cell properties in vivo is
challenging, and so considerable debate exists as to whether some proposed stem cell
populations in the adult are indeed stem cells.
Adult stem cells express transporters of the ATP-binding cassette family that actively
pump a diversity of organic molecules out of the cell. Many pharmaceuticals are
exported by these transporters conferring multidrug resistance onto the cell. This
complicates the design of drugs, for instance neural stem cell targeted therapies for
the treatment of clinical depression.
Adult stem cell research has been focused on uncovering the general molecular
mechanisms that control their self-renewal and differentiation.
Notch
The Notch pathway has been known to developmental biologists for decades. Its role
in control of stem cell proliferation has now been demonstrated for several cell types
including hematopoietic, neural, and mammary stem cells.
Wnt
These developmental pathways are also strongly implicated as stem cell regulators.
TGF
Stem cells can be used to generate healthy and functioning specialized cells, which
can then replace diseased or dysfunctional cells.
It is similar to the process of organ transplantation only the treatment consists of
transplanting cells instead of organs.

The behavior of stem cells depends on their history and on their local
context or niche.

They are not inflexible; we must simply learn how to influence them.
Single extra cellular signals that interact with cell-surface signal-detecting proteins
can direct brain and blood stem cells to specific fates. For example, immature B cells
(white blood cells) can be propagated for long periods in the presence of a cytokine
called interleukin-7.
In the absence of interleukin-7 they differentiate into mature B cells. If a key gene
transcription factor, Pax5, is missing from the immature B cells, they can be nudged
into a variety of different cell types, such as dendritic cells (antigen-presenting cells)
or macrophages (which engulf foreign material), depending on the combination of
cytokines added to the culture medium. Isolating the fewer than 20 somatic stem-cell
types, and defining their responses to external signals, will help us to decipher the
language used to build and maintain tissues.
How Does Cell Therapy Work?
Bone marrow transplants are an example of cell therapy in which the stem cells in
a donor's marrow are used to replace the blood cells of the victims of leukemia.
Cell therapy is also being used in experiments to graft new skin cells to treat
serious burn victims, and to grow new corneas for the sight-impaired.
In all of these uses, the goal is for the healthy cells to become integrated
into the body and begin to function like the patient's own cells.
Stem Cell Characteristics Make Them Good Candidates for Cell-based
Therapies
Potential to be harvested from patients
High capacity of cell proliferation in culture to obtain large number of cells from a
limited source
Ease of manipulation to replace existing non functional genes via gene transfer
methods
Ability to migrate to host's target tissues, e.g. the brain
Ability to integrate into host tissue and interact with surrounding tissue
Embryonic Stem Cells for Therapy
Advantages:
Easily available from fertility clinics
Pluripotent have ability to differentiate into cells derived from all 3 germ layers but
not the embryonic membranes
Immortal proliferate in culture & maintained in culture for several hundred
doublings
Disadvantages:
Tumorigenic any contaminating undifferentiated cells could give rise to cancer
Always allogenic immune rejection
Ethically controversial
Adult Stem Cells for Therapy
Advantages:
Can be taken form patients own cells not rejected by the immune system
Already somewhat specialized: Inducement may be simpler
Less ethical problems
Disadvantages:
Rare in mature tissues difficult to obtain in large quantities
They don't live as long in a culture as embryonic stem cells
Generally limited to differentiating into different cell types of their tissue of origin
even though plasticity may exist
Skin replacement
The knowledge of stem cells has made it possible for scientists to grow skin from a
patient's plucked hair. Skin (keratinocyte) stem cells reside in the hair follicle and can
be removed when a hair is plucked.

These cells can be cultured to form an epidermal equivalent of the patients own skin
and provides tissue for an autologous graft, bypassing the problem of rejection.
It is presently being studied in clinical trials as an alternative to surgical grafts used
for venous ulcers and burn victims
Brain cell transplantation
The identification and localization of neural stem cells, both embryonic and adult, has
been a major focus of current research.
Potential targets of neural stem cell transplants include stroke, spinal cord injury,
and neurodegenerative diseases such as Parkinson's Disease.
Stem cells can provide dopamine - a chemical lacking in victims of Parkinson's Disease
Over 250 patients have already been transplanted with human fetal tissue
Treatment of diabetes
Recently, insulin expressing cells from mouse stem cells have been generated. In
addition, the cells self assemble to form structures, which closely resemble normal
pancreatic islets and produce insulin
Future research will need to investigate how to optimize conditions for insulin
production with the aim of providing a stem cell-based therapy to treat diabetes to
replace the constant need for insulin injections
Osteocel
1st commercial product in US: bone matrix containing allogeneic human mesenchymal
stem cells
Launched in 2005 by Osiris Therapeutics
Osiris ability to supply Osteocel is limited, so it is currently marketed only for use in
certain spinal surgeries requiring bone fusion, but more extensive bone-regenerating
applications are envisaged
At May 2005 report from Swiss investment company New Venturetec, Osiris largest
backer, estimated the company could be worth $2m in revenue (all from Osteocel) in
that year rising to $10m by 2006 and peaking at about $50m in 2009 because of
supply constraints
Other brands: Chondrogen, Tissue Repair
Two of the worlds most successful blockbuster biotechnology products Epogen &
Neupogen were discovered & developed through the use of in vitro assays involving
blood stem cell technologies
Stem cells which can be expanded ex vivo and differentiated into specialized cell
types could enable the development of high-throughput screens for testing the effects
and possible toxicity of a range of drugs early in the drug development pipeline
In clinical trials at Moorfields Eye Hospital in London, surgeons restored eye sight for
six patients who lost their sight after chemical accidents and genetic diseases. The
patients went under successful stem-cell transplant.
The treatment is known as limbal stem cell therapy, and the patients who received
the treatment suffered from chemical burn or genetic disease know as aniridia
By replacing the limbal stem cells, the cornea begins to clear up as the cells are
replaced with the healthy transparent layer again.
Transgenic animals are animals (most commonly mice) that have had a foreign
gene deliberately inserted into their genome. Suchanimals are most commonly
created by the micro-injection of DNA into the pronuclei of a fertilised egg which is
subsequently implanted into the oviduct of a pseudopregnant surrogate mother.
Transgenesis is the insertion of foreign (exogenous) genes into the genome of an
organism.
The Transgene is the foreign gene that is transferred to the recipient cell/organism.
The Rationale for creation of Transgenic Animals includes:
Improve the commercial value of farm animals
Pharming. The use of farm animals as bioreactors that produce pharmaceuticals or
organs for xenotransplant.

Create mouse models for human disease

Replicate by inserting their DNA into a host cell
Gene therapy can use this to insert genes that encode for a desired protein to create
the desired trait
Four different types of viruses are usually used for human gene therapy
Creats double stranded DNA copies from RNA genome
The retrovirus goes through reverse transcription using reverse transcriptase and RNA
the double stranded viral genome integrates into the human genome using integrase
integrase inserts the gene anywhere because it has no specific site
May cause insertional mutagenesis
One gene disrupts another genes code (disrupted cell division causes cancer from
uncontrolled cell division)
vectors used are derived from the human immunodeficiency virus (HIV) and are being
evaluated for safety
Are double stranded DNA genome that cause respiratory, intestinal, and eye
infections in humans
The inserted DNA is not incorporate into genome
Not replicated though
Has to be reinserted when more cells divide
Adeno-associated Virus- small, single stranded DNA that insert genetic material at a
specific point on chromosome 19
From parvovirus family- causes no known disease and doesn't trigger patient immune
response.
Low information capacity
gene is always "on" so the protein is always being expressed, possibly even in
instances when it isn't needed.
hemophilia treatments, for example, a gene-carrying vector could be injected into a
muscle, prompting the muscle cells to produce Factor IX and thus prevent bleeding.
Study by Wilson and Kathy High (University of Pennsylvania), patients have not
needed Factor IX injections for more than a year
Double stranded DNA viruses that infect neurons
Ex. Herpes simplex virus type 1
They can introduce genes into dividing and non- dividing cells of nervous system,
which are resistant to other vectors.
Limited DNA capacity
Expression of viral genes
Initiation of anti- viral Immune response
Reversion to a replication competent state
Decreasing expression overtime.
Adenovirus
Infects many cell types
Does not integrate into host genome and can be lost.
Retrovirus
Integrates into host genome and cannot be lost
Integrates into host genome and can cause cancer
Adeno-Associated Virus (AAV)
Integrates into host genome and cannot be lost
Difficult to work with.
Herpes Simplex Virus (HSV)
DNA stays in nucleus without integrating into host genome.
Only infects cells of the nervous system.
SV 40 vectors:
SV40 is an abbreviation for Simian vacuolating virus 40 or Simian virus 40, a
polyomavirus that is found in both monkeys and humans

The pRL-SV40 Vector contains the SV40 enhancer/promoter region, which provides
strong, constitutive expression of Rluc in a variety of cell types. The vectors also
contains the SV40 origin of replication, which allows transient, episomal replication in
cells expressing the SV40 large T antigen, such as COS-1 or COS-7 cells
Downstream of the SV40 enhancer/promoter region of the pRL-SV40 Vector is a
chimeric intron comprised of the 5-donor splice site from the first intron of the
human -globin gene, and the branch and 3-acceptor splice site from an intron
preceding an immunoglobulin gene heavy chain variable region . The sequences of
the donor and acceptor splice sites, along with the branch point site, have been
modified to match the consensus sequences for optimal splicing .
Transfection studies have demonstrated that the presence of an intron flanking a
cDNA insert frequently increases the level of gene expression
In the pRL-SV40 Vector, the intron is positioned 5 to Rluc to minimize the utilization
of cryptic 5-donor splice sites that may reside within the reporter gene sequence
A T7 promoter is located downstream of the chimeric intron, immediately preceding
the Rluc reporter gene. This T7 promoter can be used to synthesize RNA transcripts in
vitro using T7 RNA Polymerase.
Renilla Luciferase Reporter Gene (Rluc) :The Renilla luciferase cDNA inserted into all of
the pRL Vectors is derived from the anthozoan coelenterate Renilla reniformis (1) but
contains nucleotide changes that were engineered during the construction of the
individual vectors. The following bases were altered in the pRL-SV40 Vector: base 924
(TC) to eliminate an internal BamH I site; base 1500 (CT) to eliminate internal Nar
I, Kas K, Ban K and Acy I sites. These nucleotide substitutions do not alter the amino
acid sequence of the encoded Renilla luciferase reporter enzyme.
SV40 Late Polyadenylation Signal: Polyadenylation signals cause the termination of
transcription by RNA polymerase II and signal the addition of approximately 200250
adenosine residues to the 3-end of the RNA transcript.
Chemical-based transfection
Chemical-based transfection can be divided into several kinds: cyclodextrin,
polymers, liposomes, or nanoparticles .
One of the cheapest methods uses calcium phosphate, originally discovered by F. L.
Graham and A. J. van der Eb in 1973
HEPES-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 By a
process not entirely understood, the cells take up some of the precipitate, and with it,
the DNA.
Other methods use highly branched organic compounds, so-called dendrimers, to
bind the DNA and get it into the cell.
Another method is the use of cationic polymers such as DEAEdextran or polyethylenimine. The negatively charged DNA binds to the polycation and
the complex is taken up by the cell via endocytosis.
Lipofection:
Lipofection (or liposome transfection) is a technique used to inject genetic material
into a cell by means of liposomes, which are vesicles that can easily merge with
the cell membrane since they are both made of a phospholipid bilayer.
Lipofection generally uses a positively charged (cationic) lipid to form an aggregate
with the negatively charged (anionic) genetic material.
A net positive charge on this aggregate has been assumed to increase the
effectiveness of transfection through the negatively charged phospholipid bilayer
Non chemical methods

Electroporation (Gene electrotransfer) is a popular method, where transient

increase in the permeability of cell membrane is achieved when the cells are exposed
to short pulses of an intense electric field.(300-400 V for 2-4 ms).
Similarly, transfection applying sonic forces to cells, referred as Sono-poration.
Optical transfection is a method where a tiny (~1 m diameter) hole is transiently
generated in the plasma membrane of a cell using a highly focused laser. This
technique was first described in 1984 by Tsukakoshi et al. In this technique, one cell at
a time is treated, making it particularly useful for single cell analysis.
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 method
A direct approach to transfection is the gene gun, where the DNA is coupled to
a nanoparticles of an inert solid (commonly gold) which is then "shot" directly into the
target cell's nucleus.
Magnetofection, or Magnet assisted transfection is a transfection method, which 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 towards and into the target cells, where the cargo is released
Microinjection refers to the process of using a glass micropipette to inject a liquid
substance at a microscopic or borderline macroscopic level. The target is often a living
cell but may also include intercellular space. Microinjection is a simple mechanical
process usually involving an inverted microscope with a magnification power of
around 200x
or processes such as cellular or pronuclear injection the target cell is positioned under
the microscope and two micromanipulators one holding the pipette and one holding
a microcapillary needle usually between 0.5 to 5 m in diameter (larger if injecting
stem cells into an embryo) are used to penetrate the cell membraneand/or
the nuclear envelope. In this way the process can be used to introduce a vector into a
single cell.
Embryonic Stem Cell approach. ES cells from a donor blastocycst are transfected
with the transgene (most likely on a plasmid).
Transfected ES cells are injected into a recipient blastocyst to form a chimera.
The chimeric blastocyct is placed into the uterus of a surrogate mother.
In vitro toxicity testing is the scientific analysis of the effects of toxic chemical
substances on cultured bacteria or mammalian cells.
In vitro (literally 'in glass') testing methods are employed primarily :
to identify potentially hazardous chemicals
to confirm the lack of certain toxic properties in the early stages of the development
of potentially useful new substances such as therapeutic drugs, agricultural chemicals
and food additives.
In vitro toxicity testing methods can be more useful and cost-effective than toxicology
studies in living animals (which are termed in vivo or "in life" methods).
Cell culture can be used to screen for toxicity both
by estimation of the basal functions of the cell (i.e. those processes common to all
types of cells) or
by tests on specialized cell functions.

For general toxicity studies, the more commonly used cell lines include the well
characterized diploid human fibroblast lines, WI-38 and tumour cell lines,
HeLa.
The first and most readily observed effect following exposure of cells to toxicants is
morphological alteration in the cell layer or cell shape in monolayer culture.
The cytotoxic concentrations of chemicals determined in vitro have been shown to
correlate well with lethal doses in laboratory animals and man for a range of selected
drugs and chemicals (Ekwall, 1983).
Morphological changes in cells exposed to chemicals (blebbing - suggesting injury of
the cell membrane or vacuolization) observed by light or electron microscopy, have
also been used to demonstrate basic cytotoxicity. (Ekwall, 1983).
These observations may provide valuable information about the pathologic processes
that occur as a consequence of exposure to a chemical substance.
Cell morphology
Blebbing, vacuolisation, fine ultrastructural modification
Cell viability
Trypan blue (enters dead cells), neutral red (actively taken up by living cells), Cr51
release
Cell growth
Cell count, plating efficiency, DNA or protein content, glucose consumption, lactate
production, NR-test, MTT-test
Metabolic parameters
O2 consumption or ATP level, pool of DNA and RNA precursors, NADH-NAD conversion.
Synthesis or release of specific molecules:
Collagen mat, heme, haemoglobin, albumin, urea, lipoprotein, bile salts,
metallothionein, glycosaminoglycans, proline and hydroxyproline, energy-dependent
choline accumulation, and histamine release .
Synthesis, activity or release of specific enzymes:
Glucuronidase, lactate-dehydrogenase, oubain-insensitive ATPase, G-6-P
dehydrogenase, glycogen phosphorylase, glutamic-oxalacetic transaminase,
glutamic-pyruvic transaminase, acetylcholinesterase, and renin.
Interactions of compound with cells:
Phagocytosis, cytoplasmic inclusions, intracellular accumulation, uptake or binding of
compound to cytosol and lipoproteins, mitogenic response.
Alterations of metabolic pathways:
Methaemoglobin reduction, glucose-transport, 5-methyl tetra hydropholate
accumulation, hormone-stimulated gluconeogenesis, lipid peroxidation, fat
accumulation and glucosamine and galactose incorporation.
Cell surface activities:
Adhesiveness, antibody-mediated rosette formation, complement deposition on
treated cell membrane, chemotactic migration, GABA-mediated postsynaptic
inhibition, membrane polarization, fibre retraction or outgrowth, and
electrophysiological alteration.
Intracellular markers
Mitochondria, lysosomes, peroxisomes
Cell viability and Cytotoxicity assays are used for drug screening and cytotoxicity tests
of chemicals.
This in vitro test evaluates the potential of the materials or their extracts to cause
damage to cells in culture.(useful in evaluating the toxicity or irritancy potential of
materials and chemicals. )
Cell cultures are suitable test systems for the determination of cytotoxic reactions
such as changes in cell cycle, inhibition of cell division, and cell death. (caused by
eluates or extracts of products either natural or processed.)
A. Qualitative Cytotoxicity Tests

Three different qualitative cytotoxicity tests are commonly used :

1. Extraction method/ MEM Elution
2. Agar Diffusion or Agarose overlay assay
3. Direct contact method
In general, in these tests, toxicity is verified after a period of exposure (typically 2472
hours) of the cells to the extract or device.
MEM Elution - Test on Extracts (ISO 10993-5)
The test material is extracted for 24 hours in Minimum Essential Medium (MEM).
An extract is prepared from the test material which is then placed over the cultured
cells. (L-929 mouse fibroblast cells)
Following incubation, the cells are examined microscopically for morphological
changes, degeneration and lysis of the cells.
Potentially cytotoxic substances are uniformly distributed throughout the cell culture
Agar Diffusion or Agarose overlay assay - (ISO 10993-5)
In this method, a thin layer of nutrient-supplemented agar is placed over the cultured
cells.(L-929 mouse fibroblast cells )
The test material (or an extract of the test material dried on filter paper/sample) is
placed on top of the agar layer, and the cells are incubated for 24 hours.
Cytotoxic leachates diffuse into the cell layer via the agar, and a zone of malformed,
degenerative or lysed cells under and around the test material indicates cytotoxicity.
MRC-5 Human Embryonic Lung Cells
Direct Contact (ISO 10993-5)
In this method, a piece of test material is placed directly onto cells growing on culture
medium.(without the agar layer)
Cell cultures are grown to a standard monolayer.
The cells are then incubated for 24 hours at 37C.
During incubation, leachable chemicals in the test material can diffuse into the culture
medium and contact the cell layer.
Subsequently, the monolayers are examined microscopically for the presence of
morphological changes, reduction in cell density or lysis of cells around the test
material.
MTT assay is used often in determining cell viability (detects viable cells).
The MTT is a colorimetric method that measures the reduction of yellow 3-(4,5dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide into an insoluble
purple formazan product by mitochondrial succinate dehydrogenase.
MTT being water soluble can penetrate through cell membrane, water insoluble
formazan is trapped inside the cell.
Dead cells do not have active mitochondrial reductases (as the cellular reduction is
only catalyzed by living cells), MTT is not reduced and the purple formazan is not
formed.
Samples are read using an ELISA plate reader at a wavelength of 570 nm.
The amount of color produced is directly proportional to the number of viable cells.
The MTT can be used to evaluate the cytotoxicity of:
Toxic compounds
Toxins and environmental pollutants
Potential anti-cancer drugs
Antibodies to examine growth inhibiting potential
The acetomethoxy derivate of calcein (calcein AM) is used in testing of cell viability
as it can be transported through the cellular membrane into live cells.
After transport into the cells, intracellular esterases remove the acetomethoxy group,
the molecule gets trapped inside and gives out strong green fluorescence.
As dead cells lack active esterases, only live cells are labeled and counted by flow
cytometry.
Lysosomal activity

Membrane permiability
The neutral red (NR) assay is a cell survival chemo sensitivity assay.
This assay is based on the incorporation of NR into the lysosomes of viable cells after
being incubation with test agents.
NR (3-amino-7-dimethyl-2-methylphenazine hydrochloride) is a weak cationic dye that
readily penetrates cell membranes by non-ionic diffusion, accumulating intracelluarly
in lysosomes, where it binds with anionic sites in the lysosmal matrix.
Therefore, it is possible to distinguish between viable, damaged or dead cells as viable
cells take up the NR dye, damaged or dead cells do not.
When a reduced cytotoxic effect was determined by neutral red (NR-test), which
shows the activity of lysosomal enzymes, microscopically multiplication or
enlargement of lysosomes was observed.
Lactate dehydrogenase (LDH), which is a soluble cytosolic enzyme present in most
eukaryotic cells, releases into culture medium upon cell death due to damage of
plasma membrane.
The increase of the LDH activity in culture supernatant is proportional to the number
of lysed cells.
LDH activity, therefore, can be used as an indicator of cell membrane integrity and
serves as a general means to assess cytotoxicity resulting from chemical compounds
or environmental toxic factors.
LDH Assay measures LDH activity present in the culture medium using a coupled twostep reaction.
In the first step, LDH catalyzes the reduction of NAD+ to NADH and H+ by oxidation of
lactate to pyruvate.
In the second step of the reaction, diaphorase (NADPH dehydrogenase) uses the
newly-formed NADH and H+ to catalyze the reduction of a tetrazolium salt to
highly-colored formazan which absorbs strongly at 490-520 nm.
A vaccine is a biological preparation that improves immunity to a particular disease.
A vaccine typically contains an agent that resembles a disease-causing
microorganism, and is often made from weakened or killed forms of the microbe. The
agent stimulates the body's immune system to recognize the agent as foreign,
destroy it, and "remember" it, so that the immune system can more easily recognize
and destroy any of these microorganisms that it later encounters
Establish resistance to virus/pathological organism by evoking an immune response
Give host a foreign organism/protein in non-infectious form
Antibodies are generated Ab binds to surface proteins of organism
.Types
Inactivated (Killed)
Live
Attentuated (Live, Non-infectious)
LIVE MORE EFFECTIVE THAN KILLED
II. Pathogens
Bacteria
Virus
Parasites
Attenuated live microbe (usually virus) which has a vital function inactivated by
heat, chemicals or genetic manipulation
e.g. Rabies virus vaccine, MMR (Measles, Mumps and Rubella)
BCG (Bacillus Calmette Guerin vaccine for Mycobacterium tuberculosis
Risk it could revert back to infectious agent
will stimulate both cell mediated and antibody mediated immune responses
Inactivated uses toxoid inactivated toxins which are purified proteins
stimulates the antibody mediated response only
e.g. DPT (diphtheria, pertussis, tetanus toxoids)

stimulates the antibody mediated response only

Component (subunit) contains purified components from bacteria and viruses
How recombinant viruses are made
Hepatitis B vaccine purified viral coat protein
Streptococcus pneumoniae (PneumoShot) capsular polysaccharide
Hemophilus influenzae (HiB) capsular polysaccharide, part of DPTPolio- Hib vaccine
given to infants
Nesseria meningiditis capsular polysaccharide: stimulates the antibody mediated
response only
Booster Shots: same vaccine given at a later date (e.g. DT given every 10 years
to refresh the memory cell population
Adjuvant: chemicals in the vaccine solution that enhance the immune response
Alum Ag in the vaccine clumps with the alum such that the Ag is released
slowly, like a time-release capsule
gives more time for memory cells to form
cant grow all organisms in culture
safety to lab personnel
Expense
insufficient attentuation
reversion to infectious state
need refrigeration
do not work for all infectious agents
infants/children receive them immature immunity
Subunit Vaccines
peptide vaccines
Genetic immunization
Attenuated Vaccines
Vector Vaccines
Bacterial Antigen Delivery Systems
Delete Virulence Genes (can not revert)
V/B as Vaccine
Clone gene for pathogenic antigen into non-pathogenic virus or bacteria
V/B as Vaccine
Clone pathogenic antigen gene into expression vector
Vaccinate with protein
Subunit
Peptide
Do NOT use entire virus or bacteria (pathogenic agent)
Use components of pathogenic organism instead of whole organism
Advantage: no extraneous pathogenic particles ie DNA
Disadvantage: Is protein same as in situ?
Cost
HSV
Problem with Traditional vaccine- HSV is oncogenic
envelope glycoprotein D (gD) elicits Ab response
Clone gene for gD into vector
Express in mammalian cells
Transmembrane protein
modify gene to remove TM portion
Foot -and-Mouth Disease virus
cattle/pigs
VP1 capsid viral protein elicits response - used this protein as Vaccine
B TuberculosisMycobacterium tuberculosis antibiotic resistant strains use purified
extracellular (secreted) proteins as Vaccine

Use discrete portion (domain) of a surface protein as Vaccine

These domains are epitopes
antigenic determinants
are recognized by antibodies
CARRIER PROTEINS help vaccine production
Small Peptides are often Digested
Carrier Proteins Make more Stable
Problem:
Large quantities of peptide needed to be used to get immunological response
Solution:
Use highly immunogenic carrier molecule
HBcAg was a suitable carrier
(Hepatitis Core Protein)
Fused peptide DNA with gene for HBcAg
This fusion protein used as Vaccine
Delivery of a gene for the antigen to a host organism
Use vector containing cDNA from viral protein/
eukaryotic promoter
Inject into muscle/microprojectile system
POTENTIAL
eliminates purification of antigen
protein is modified post-translationally
FATE of plasmid DNA
integration?
degradation?
Cholera
caused by bacterium
lives in intestine causing diarrhea, dehydration
poor sanitation (water supply, sewage)
secretes an enterotoxin (A1) which causes disease
killed vaccine not effective long-term
subunit not effective
Phenol-killed cholera used as vaccine currently
Typically double deletions are preferred can not multiple in host
Insert tetracycline gene into bacteriums host chromosome
This gene interrupts A1 peptide gene
(toxic portion of the enterotoxin)
NOT ACCEPTABLE
Reversion by spontaneous excision
Deleted A1 peptide sequence created
Vaccinia good candidate for a live recombinant viral vaccine
benign virus
replicate in cytoplasm (viral replication genes)
easy to store
Insert cloned gene encoding antigen
Interrupt thymidine kinase (non-essential gene)
Infect host cell with native virus
D) Transform these cells with recombinant plasmid
E) HOMOLOGOUS RECOMBINATION
F) Select cells which are resistant to BROMODEOXYURIDINE
**MODIFIED VIRUS USED AS VACCINE**
Use live nonpathogenic bacterium which contains antigen
(Salmonella)
(epitope from cholera)
Insert antigen gene into flagellin gene

Epitope is expressed on the flagellum surface

***Flagellin-engineered bacteria is VACCINE**
Advantage - Oral Administration
transplantation is the moving of an organ/ Tissue/ Cells from one body to another
or from a donor site to another location on the person's own body, to replace the
recipient's damaged or absent organ.
. Auto graft or Autogenic graft,
Isograft or Syngraft or Syngenetic graft,
allografts or Homografts,
Xenograft.
When tissue is transplanted from one site to another in the same individual, the
transplant is referred as "auto graft" or "autogenic graft" (From Greek Auto=Self).
Immune system of recipient accepts the auto graft very easily, because antigens of
recipient cells and the transplanted tissue are alike.
The graft taken from a genetically identical person is known as Isograft or Syngraft or
Syngentic graft. This kind of transplantation is possible between two genetically
identical twins.
Since development of identical twins takes place from a single zygote, identical twins
share same genes that are responsible for the production of antigens.
If the transplantation is carried between genetically different members of the same
species then graft is called as "allograft". The allograft is formally named as
"Homograft".
The histo compartbility antigens of allograft are dissimilar with the host histo
compartbility antigens. Hence immune system of recipient/ host identifies the graft as
foreign and induces an immune response against to it, resulting rejection of graft.
If the transplantation between individuals of two different species is carried, for eg.
Transplanting monkey liver to human, the graft is referred as "Xenograft".
Since the histocompatibility genes are quite different, hosts body rejects the graft
more vigorously.
Somatic cell fusion or Hybrid cells can be produced by fusing different types of
somatic cells from two different tissues or species in a cell culture media. Usually
human somatic cells like fibrocyte or leucocyte are fused with continuous cell lines.
Somatic cells of different types can be fused to obtain hybrid cells. Hybrid cells are
useful in a variety of ways, e.g.,
to study the control of cell division and gene expression,
to investigate malignant transformations,
to obtain viral replication,
for gene or chromosome mapping and for
production of monoclonal antibodies by producing hybridoma (hybrid cells between an
immortalised cell and an antibody producing lymphocyte), etc.
Chromosome mapping through somatic cell hybridization is essentially based on
fusion of human and mouse somatic cells. Generally, human fibrocytes or leucocytes
are fused with mouse continuous cell lines.
In 1960s, in France for the first time, the hybrid cells were successfully produced from
mixed cultures of two different cell lines of mouse.
When somatic cells of human and mouse origin or cells of any two species of
mammals or two cells of same species are mixed, spontaneously cell fusion occurs at
very low rate.
Therefore by adding ultraviolet inactivated Sendai (parainfluenza) virus or chemical
compound known as polyethylene glycol (PEG) increases the somatic cell fusion by
100 to 1000 times.
Polyethylene glycol or Sendai virus adhere to the cell membrane and alter their
properties in such a way that, it facilitates cell fusion. Fusion of two different cells
produces a special type of cells known as heterokaryon.

That is a single hybrid cell will have two nuclei, one from each of the fused cells. After
some time, the two different nuclei fuse to form a single nucleus.
The fusogen induce two different cells first to form heterokaryons.
During mitosis, chromosomes of heterokaryon move towards the two poles and later
on fuse to form hybrids.
It is important to remove the surface carbohydrates to bring about cell fusion.
The somatic cell hybridization involves following three aspects
Fusion of Protoplasts:
As the isolated protoplasts are devoid of cell walls, there in vitro fusion becomes
relatively easy.
There are no barriers of incompatibility (at interspecific, inter-generic or even at interkingdom levels) for the protoplast fusion.
Protoplast fusion that involves mixing of protoplasts of two different genomes can be
achieved by spontaneous, mechanical, or induced fusion methods.
1..1. Spontaneous fusion:
Cell fusion is a natural process as is observed in case of egg fertilization.
During the course of enzymatic degradation of cell walls, some of the adjoining
protoplasts may fuse to form homokaryocytes (homokaryons).
These fused cells may sometimes contain high number of nuclei (2-40).
This is mainly because of expansion and subsequent coalescence of plasmodermal
connections between cells.
The frequency of homokaryon formation was found to be high in protoplasts isolated
from dividing cultured cells. Spontaneously fused protoplasts, however, cannot
regenerate into whole plants, except undergoing a few cell divisions.
Mechanical fusion:
The protoplasts can be pushed together mechanically to fuse.
Protoplasts of cells in enzyme solutions can be fused by gentle trapping in a
depression slide.
Mechanical fusion may damage protoplasts by causing injuries.
Induced fusion:
Freshly isolated protoplasts can be fused by induction.
There are several fusion-inducing agents which are collectively referred to as
fusogens e.g. NaN03, high pH/Ca2+, polyethylene glycol, polyvinyl alcohol, lysozyme,
concavalin A, dextran, dextran sulfate, fatty acids and esters, electro fusion.
Some of the fusogens and their use in induced fusion are described
1.3.1 Treatment with sodium nitrate:
The isolated protoplasts are exposed to a mixture of 5.5% NaNO3 in 10% sucrose
solution.
Incubation is carried out for 5 minutes at 35C, followed by centrifugation (200 x g for
5 min).
The protoplast pellet is kept in a water bath at 30C for about 30 minutes, during
which period protoplast fusion occurs. NaNO3 treatment results in a low frequency of
heterokaryon formation.
1.3.2. High pH and high Ca2+ ion treatment:
This method was first used for the fusion of tobacco protoplasts, and is now in use for
several plants / animals cells.
The method consists of incubating protoplasts in a solution of 0.4 M mannitol
containing 0.05 M CaCI2 at pH 10.5 (glycine-NaOH buffer) and temperature 3 7C for
30-40 minutes.
The protoplasts form aggregates, and fusion usually occurs within 10 minutes.
By this method, 20-50% of the protoplasts are involved in fusion.
1.3.3 Polyethylene glycol (PEG) treatment:
This has become the method of choice, due to its high success rate, for the fusion of
protoplasts from many plant species.

The isolated protoplasts in culture medium (1 ml) are mixed with equal volume (1 ml)
of 28-56% PEG (mol. wt. 1500-6000 Daltons) in a tube.
PEG enhances fusion of protoplasts in several species.
This tube is shaken and then allowed to settle. The settled protoplasts are washed
several times with culture medium.
PEG treatment method is widely used protoplast fusion as it has several
advantages:
It results in a reproducible high-frequency of heterokaryon formation.
Low toxicity to cells.
Reduced formation of bi-nucleate heterokaryons.
PEG-induced fusion is non-specific and therefore can be used for a wide range of cells.
1.3.4. Electro-fusion:
In this method, electrical field is used for protoplast fusion.
When the protoplasts are placed in a culture vessel fitted with micro- electrodes and
an electrical shock is applied, protoplasts are induced to fuse.
Electro-fusion technique is simple, quick and efficient and hence preferred by many
workers.
Further, the cells formed due to electro-fusion do not show cytotoxic responses as is
the case with the use of fusogens (including PEG).
The major limitation of this method is the requirement of specialized and costly
equipment.
The fusion of protoplasts involves three phases agglutination, plasma membrane
fusion and formation of heterokaryons.
1. Agglutination (adhesion):
When two protoplasts are in close contact with each other, adhesion occurs.
Agglutination can be induced by fusogens e.g. PEG, high pH and high Ca2+.
2. Plasma membrane fusion:
Protoplast membranes get fused at localized sites at the points of adhesion. This leads
to the formation of cytoplasmic bridges between protoplasts. The plasma membrane
fusion can be increased by high pH and high Ca2+, high temperature and PEG, as
explained below.
(a) High pH and high Ca2+ ions neutralize the surface charges on the protoplasts. This
allows closer contact and membrane fusion between agglutinated protoplasts.
(b) High temperature helps in the intermingling of lipid molecules of agglutinated
protoplast membranes so that membrane fusion occurs.
(c) PEG causes rapid agglutination and formation of clumps of protoplasts. This results
in the formation of tight adhesions of membranes and consequently their fusion.
3. Formation of heterokaryons:
The fused protoplasts get rounded as a result of cytoplasmic bridges leading to the
formation of spherical homokaryon or heterokaryon.
About 20-25% of the protoplasts are actually involved in the fusion.
After the fusion process, the protoplast population consists of a heterogenous mixture
of un-fused chloroplasts, homokaryons and heterokaryons.
It is therefore necessary to select the hybrid cells (heterokaryons).
The commonly used methods employed for the selection of hybrid cells are
biochemical, visual and cytometric methods.
Biochemical methods:
The biochemical methods for selection of hybrid cells are based on the use of
biochemical compounds in the medium (selection medium).
These compounds help to sort out the hybrid and parental cells based on their
differences in the expression of characters.
1.a. Drug sensitivity:
This method is useful for the selection hybrids of two cells/ species, if one of them is
sensitive to a drug.

Drug sensitivity technique was originally developed by Power et al (1976) for the
selection of hybrids of Petunia sp.
When these two species are fused, the fused protoplasts derive both the characters
formation of macroscopic colonies and resistance to actinomycin D on selection
medium.
This helps in the selection of hybrids, as the parental protoplasts of both the species
fail to grow.
1.b. Auxotrophic mutants:
Auxotrophs are mutants that cannot grow on a minimal medium and therefore require
specific compounds to be added to the medium.
Nitrate reductase deficient mutants of tobacco (N. tabacum) are known.
The parental protoplasts of such species cannot grow with nitrate as the sole source
of nitrogen while the hybrids can grow.
Two species of nitrate reductase deficiency one due to lack of apoenzyme (nia-type
mutant) and the other due to lack of molybdenum cofactor (cnx- type mutant) are
known.
The parental protoplasts cannot grow on nitrate medium while the hybrid protoplasts
can grow
1.C. Visual methods:
Visual selection of hybrid cells, although tedious is very efficient. In some of the
somatic hybridization experiments, chloroplast deficient (albino or non-green)
protoplasts of one parent are fused with green protoplasts of another parent. This
facilitates the visual identification of haterokaryons under light microscope. The
heterokaryons are bigger and green in colour while the parental protoplasts are either
small or colourless. Further identification of these heterokaryons has to be carried out
to develop the specific hybrid plant.
There are two approaches in this direction growth on selection medium, and
mechanical isolation.
1.C.1. Visual selection coupled with differential media growth:
There exist certain natural differences in the sensitivity of protoplasts to the nutrients
of a given medium. Thus, some media can selectively support the development of
hybrids but not the parental protoplasts. A diagrammatic representation of visual
selection coupled with the growth of heterokaryons on a selection medium is given in
Fig
The HAT medium is most commonly used selective media. This selective media is
used for the selection of hybrid cells. This selective medium contains hypoxanthine,
aminopterin and thymidine, hence the name. Aminopterin stops the biosynthesis of
purines and pyrimidines using simple sugars and also amino acids.
2. However normal human and mouse cells still survive and replicate using
hypoxanthine and thymidine which are present in the medium via salvage pathway.
3. Hypoxanthine present in the medium is converted into guanine by the help of an
enzyme known as enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT),
phosphorylation of thymidine is done by an enzyme known as thymidine kinase (TK).
4. Only cells with active hypoxanthine guanine phosphoribosyltransferase and
thymidine kinase enzymes can survive and proliferate on a HAT medium. The cells
with no active form of these enzymes will not divide in the HAT medium.
5. To use HAT medium as selective media, used human somatic cells must be deficient
for any one of the two enzymes. While selected mouse cell must be deficient for the
other enzyme. For example hypoxanthine guanine phosphoribosyltransferase deficient
human somatic cells are fused with thymidine kinase deficient mouse cell.
6. Fusion of these somatic cells will produce hybrid cells with both the enzymes such
as thymidine kinase and hypoxanthine guanine phosphoribosyltransferase and hybrid
cells will proliferate on the HAT medium, whereas the human somatic cell and mouse
somatic cell will not proliferate.

1.C. 2. Mechanical isolation:

The visually identified heterokaryons under the microscope can be isolated by
mechanical means. This involves the use of a special pipette namely Drummond
pipette. The so isolated heterokaryons can be cloned to finally produce somatic hybrid
plants. The major limitation of this method is that each type of hybrid cell requires a
special culture medium for its growth. This can be overcome by employing micro drop
culture of single cells using feeder layers .
2. Cytometry methods:
Some workers use flow cytometry and fluorescent-activated cell sorting techniques for
the analysis of plant protoplasts while their viability is maintained. The same
techniques can also be applied for sorting and selection of heterokaryons. The hybrid
cells derived from such selections have proved useful for the development of certain
somatic hybrid plants.
The development of hybrid cells followed by the generation of hybrid plants animals is
a clear proof of genetic contribution from both the parental protoplasts. The hybridity
must be established only from euploid and not from aneuploid hybrids. Some of the
commonly used approaches for the identification of hybrid plants are briefly
described.
1. Morphology of hybrid plants:
Morphological features of hybrid cells which usually are intermediate between two
parents can be identified. These include shape, cell area, membrane morphology, its
structure, size and color, and organellar morphology.
The somatic hybrids such as pomatoes and topatoes which are the fused products of
potato and tomato show abnormal morphology, and thus can be identified. Although
the genetic basis of the morphological characters has not been clearly known,
intermediate morphological features suggest that the traits are under the control of
multiple genes. It is preferable to support hybrid morphological characters with
evidence of genetic data.
2. Isoenzyme analysis of hybrid plants:
The multiple forms of an enzyme catalysing the same reaction are referred to as
isoenzymes. Electrophoretic patterns of isoenzymes have been widely used to verify
hybridity. Somatic hybrids possess specific isoenzymes (of certain enzymes) of one or
the other parent or both the parents simultaneously.
There are many enzymes possessing unique isoenzymes that can be used for the
identification of somatic hybrids e.g. amylase, esterase, aspartate aminotransferase,
phosphodiesterase, isoperoxidase, and hydrogenases (of alcohol, lactate, malate). If
the enzyme is dimeric (having two subunits), somatic hybrids usually contain an
isoenzyme with an intermediate mobility properties.
3. Chromosomal constitution:
The number of chromosomes present in the hybrid cells can be directly counted. This
provides information on the ploidy state of the cells. The somatic hybrids are expected
to possess chromosomes that are equal to the total number of chromosomes
originally present in the parental protoplasts. Sometimes, the hybrids are found to
contain more chromosomes than the total of both the parents. The presence of
chromosomal markers is greatly useful for the genetic analysis of hybrid cells.
If the chromosome number in the hybrid is the sum of the chromosomes of the two
parental protoplasts, the hybrid is said to be symmetric.
Symmetric hybrids between incompatible species are usually sterile. This may be due
to production of 3n hybrids by fusing 2n of one species with n of another species.
Asymmetric hybrids have abnormal or wide variations in the chromosome number
than the exact total of two species.
These hybrids are usually formatted with full somatic complement of one parental
species while all or nearly all of the chromosomes of other parental species are lost
during mitotic divisions.

Asymmetric hybrids may be regarded as cybrids but for the introgressed genes.
Cybrids:
The cytoplasmic hybrids where the nucleus is derived from only one parent and the
cytoplasm is derived from both the parents are referred to as cybrids. The
phenomenon of formation of cybrids is regarded as cybridization. Normally, cybrids
are produced when protoplasts from two phytogenetically distinct species are fused.
Genetically, cybrids are hybrids only for cytoplasmic traits.
These are some of the applications of hybrid cells:
1. Hybrid
2. Hybrid
3. Hybrid
cells.
4. Hybrid
5. Hybrid
6. Hybrid

cells are used to study the gene expression.

cells are used to study the basics of cell division
cells can be used to study the transformation of normal cells into malignant
cells are used to obtain viral replication
cells are used for chromosome or gene mapping
cells are also used in the production of monoclonal antibodies