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Genetic Recombination
205
PROTOPLAST FUSION
Protoplast fusion is a physical phenomenon wherein two or more protoplasts come in contact
and adhere with one another either spontaneously, or in presence of fusion inducing agents. The
protoplast is a cell of which cell wall is removed. The cytoplasmic membrane is the outermost
layer in such a cell. Specific lytic enzymes are used to remove cell wall and obtain protoplast. By
protoplast fusion, it is possible to transfer some useful genes, such as genes for disease resistance,
frost hardiness, nitrogen fixation, rapid growth rate, more product formation rate, drought resistance,
protein quality, herbicide resistance and heat and cold resistance from one species to another.
Protoplast fusion is an important tool in strain improvement for bringing genetic recombinations
and developing hybrid strains in filamentous fungi.
Protoplast fusion is used to combine genes from different organisms to create strains with
desired properties. This technique is more useful in engineering of microbial strains for desirable
industrial properties.
Enzymes Used for Breaking of Cell Walls
For protoplast fusion, it is mandatory that the cell wall of plant and microorganisms is degraded.
Various enzymes are used for this process. The enzymes cellulase and pectinase or macerozyme
acting on plant cell wall. Bacterial cell walls are degraded by the action of lysozyme. Fungal wall is
degraded by Novozyme 234 that includes glucanase and chitinase. Streptomyces cell wall degraded
by action of lysozyme and achromopeptidase is another example.
Methods of Protoplast Fusion
Different techniques of protoplast fusion are in vogue.
1. Spontaneous fusion: It is the phenomena wherein the protoplast during isolation often fuses
spontaneously. During the enzyme treatment, protoplasts from adjoining cells fuse through
their plasmodesmata to form multinucleate protoplasts.
2. Induced fusion: Fusion of freely isolated protoplasts from different sources with the help of
fusion inducing chemical agents is known as induced fusion. Normally, isolated protoplasts
do not fuse with each other, because the surface of isolated protoplast carries negative charges
(<10 mV to <30 mV) around the plasma membrane. There is a strong tendency witnessed in
the protoplasts to repel each other due to same charges. This type of fusion needs a fusion-
inducing chemical that actually reduces the electronegativity of the isolated protoplast and
allows it to fuse with each others. The isolated protoplast can be then induced to fuse by
adopting any one technique.
3. Mechanical fusion: In this process, the isolated protoplast is brought into intimate physical
contact mechanically under microscope using micromanipulator or perfusion micropipette.
4. Chemofusion: It is a nonspecific and inexpensive technique. It can cause massive fusion
product. It can be cytotoxic and nonselective and possesses less fusion frequency. Several
Textbook of Pharmaceutical Biotechnology
206
chemicals are used to induce protoplast fusion, such as PEG, sodium nitrate and calcium,
calcium ions (Ca
++
).
5. Electrofusion: Mild electric stimulation is used to fuse protoplast. In this two glass capillary
microelectrodes are placed in contact with the protoplast. An electric field of low strength
(10 kvm
<1
) gives rise to dielectrophoretic dipole generation within the protoplast suspension
that leads to pearl chain arrangement of protoplasts. Subsequent application of high strength
of electric fields (100 kvm
<1
) for some microseconds results in electric breakdown of membrane
and subsequent fusion.
Protoplast Fusion in Plants
The somatic fusion is achieved in the following steps:
1. The removal of the cell wall of one cell of each type of plant using cellulase enzyme to
produce a somatic cell called protoplast.
2. The cells are then fused using both an electric shock (electrofusion) to join the cells and
the nuclei fused together or by chemical treatment. The resulting fused nucleus is called
heterokaryon.
3. The somatic hybrid cell has its cell wall induced to form using hormones.
4. The cells are then grown into calluses that are then further grown to plantlets and finally to a
full plant known as a somatic hybrid.
Protoplast Fusion in Fungi
Production and regeneration of protoplasts is a useful technique for fungal transformations.
Commercial preparation of enzymes that contain mixture of products to digest fungal cell wall is
used for protoplast fusion. Novozyme 234 includes enzyme mixture of glucanases and chitinaze.
It is added to rapidly growing fungal tissue suspended in an osmotic buffer (e.g. 0.6 mol
<
KCl,
1.2 mol
<
sorbitol or 1.2 mol
<
MgSO
4
). The protoplasts and DNA are mixed in the presence of
15% (w/v) PEG 6000 and pH buffer (TRIS-HCl). Around 10 mL
<
PEG causes clump formation in
protoplasts. The mycelium is grown on cellophane at 37°C and placed on agar overnight. It is then
incubated with enzyme at 30°C for 1.5 h in empty Petri dish containing KCl. The protoplasts are
then filtered and washed in KCl, centrifuged and resuspended.
Protoplast Technology for Streptomyces spp.
For obtaining protoplasts from Streptomyces, lysozyme is used, which breaks glycan portion of
peptidoglycan wall. The cultures from spore suspension (2 days in shaker at 30°C) are harvested by
centrifugation, resuspended in 0.03 mol
<1
sucrose, washed and finally reharvested and resuspended
in lysozyme solution in protoplasting medium (30 min; 2 h at 30°C).
Protoplast Fusion in Bacteria
In Gram-positive bacteria, protoplast fusion can be carried out at low frequency. For Gram-negative
bacteria, it is possible to obtain protoplasts, but regeneration is difficult.
Genetic Recombination
207
GENE CLONING
Cloning
The word clone is used in many different contexts in biological research. In its most simple and
strict sense, it refers to a precise genetic copy of a molecule, cell, plant, animal or human being.
Cloning refers to established technologies that have been part of agricultural practice for a very
long time and currently form an important part of the foundations of modern biological research.
The genetically identical copies of whole organisms are common. In the plant breeding
world they are commonly referred to as ‘varieties’ rather than clones. Many valuable horticultural
or agricultural strains are maintained solely by vegetative propagation from an original plant,
reflecting the ease with which it is possible to regenerate a complete plant from a small cutting.
The developmental process in animals does not usually permit cloning, as easily as, in plants. Many
simpler invertebrate species, such as certain kinds of worms, are capable of regenerating a whole
organism from a small piece, even though this is not necessarily their usual mode of reproduction.
The vertebrates have lost this ability entirely, although regeneration of certain limbs, organs or
tissues can occur to varying degrees in some animals.
A single adult vertebrate cannot generate another whole organism. The cloning of vertebrates
does occur in nature, in a limited way. The twins occur by chance, in humans and other mammals
with the separation of a single embryo into halves at an early stage of development. The resulting
offsprings are genetically identical, having been derived from one zygote that resulted from the
fertilization of one egg by one sperm.
It is a process that gives rise to genetically identical organisms. Cloning of higher organisms leads
to duplicity or creation of identical twins. It is an asexual reproduction ( replication) of mammals. In
mammalian cloning, DNA of adult cell is injected into oocyte or unfertilized egg cell whose DNA
is removed. So, in this process, DNAs from two different sources are not combined. Instead, DNA
from one source combines with DNA (cell) regulating substances of other cell. The special regulatory
(chemical) substances present in oocyte reprogrammed DNA, so that whole organism can develop
from the DNA. Thus, the egg cell develops into embryo instead of adult cell. Later, these embryos are
transferred to foster mothers. Finally, foster mother delivers baby lamb identical to donor or adult cell.
Gene Targeting
It is a modified form of the cloning technique. It involves modification or manipulation of somatic cell
DNA before it is injected into enucleated oocyte. It involves homologous recombination of target gene
and host DNA. Somatic cell is first transformed, transfected with gene (DNA) of interest, which is
meant for gene targeting. The rest of the technique is identical to that of cloning gene targeting.
1. Gene knockout: It involves targeted inactivation of gene. The mutations are introduced into
somatic cell DNA to make gene inactive. This DNA is later expressed in oocyte. By this, the
clones produced lacks gene that is inactivated.
2. Gene knockin: It involves targeted insertion of gene. Gene is expressed in clones.
Textbook of Pharmaceutical Biotechnology
208
A gene of interest is inserted into somatic DNA before it is injected into enucleated oocyte.
Applications
1. Gene targeting is used to study genetics of human diseases and gene therapy. In gene therapy,
inactive copy of gene is replaced with functional gene.
2. This can be applied in any animal species that allows selective breeding of animals for
generation.
3. This method is successfully done in mice, sheep and flies.
4. This is used to establish function of gene or gene product, such as enzyme or hormone, etc.
5. In pharmaceutical industry, gene targeting is used to overexpress proteins of therapeutic value
in milch animals.
Stem Cells
Isolation of human stem cells by using two different approaches was reported in 1998. These
cells are able to differentiate into all types of cells or tissues. Further, they can be maintained as
undifferentiated cells in culture and are able to reproduce themselves throughout the lifespan of
organisms.
The stems cells exist in growing and adult humans. Stem cells are isolated from embryo, cord
blood, bone marrow, liver, brain, etc. Only ES cells are able to differentiate into any cell type.
Others produce only narrow range of cells. Generally the developmental potential of a stem cell is
restricted to differentiate cells of the tissue in which they are present.
Applications
Stem cells have many potential uses in medicine and molecular biology.
1. They are used for replacement of lost or degenerated tissue.
2. They are used in gene therapy, e.g. neural stem cells are used for gene therapy of tumour
growth suppression.
3. They are useful in delivering therapeutic gene products directly into the tissue.
4. Genetically engineered stem cells express therapeutic genes in the tissue of choice.
5. They are useful for exploring normal process of tissue development.
DEVELOPMENT OF HYBRIDOMA BY MONOCLONAL ANTIBODIES
Hybridoma Technology
Hybridoma technology involves the preparation of hybridoma ( hybrid) cells to produce monoclonal
antibodies. Monoclonal antibodies are antibodies produced by one cell line (clone). They are directed
against one specific antigen. The lymphocytes in the body are polyclonal (multiple cell lines), and
they can produce many types of antibodies ( polyclonal) against antigens. Separation of single cell
line that produces only one antibody from the mixture of polyclonal cells is a hard task.
Genetic Recombination
209
Hybridomas
A simple method of producing single cell line of polyclonal cells involves fusion of two cell
populations. Fusion generates hybrid cells with desired properties. These hybridoma cells produce
monoclonal antibodies.
Different steps involved in hybridoma technology are summarized below.
1. A mouse is immunized with an antigen.
2. The spleen of the mouse is removed after several weeks. During this period, antibody-
producing cells are formed in sufficient amounts in the spleen.
3. The B lymphocytes are removed from spleen and fused with myeloma cells in the presence
of PEG. Myeloma cells are used for fusion because lymphocytes obtained after immunization
are incapable of continuous growth in culture. The fusion of lymphocytes with myeloma
cells leads to immortalization (infinite lifespan) of cells. Moreover, the myeloma cells lack
HGPRTase that is used as genetic marker to identify fused cells.
4. The fused cells are allowed to grow in medium containing hypoxanthine-aminopterin-
thymidine ( HAT).
5. The spleen cells die in the medium because they are not immortalized, and myeloma cells also
die because they cannot survive in HAT medium due to lack of HGPRTase.
6. Only hybridoma cells formed by the fusion of lymphocytes with myeloma cells proliferate,
because they are immortal and contain HGPRTase of lymphocytes. Thus, the hybridoma cells are
obtained. Further, the DNA of hybridoma cells is derived from myeloma cells and lymphocytes.
The hybridoma cells are screened for monoclonal cells by using antibodies (Fig. 13.5).
Hybridization Techniques
These techniques are based on the tendency of two DNA strands to form duplex. Interstrand
hydrogen bonding between complementary sequences favours duplex formation. A DNA strand and
RNA strand containing complementary sequences can also form duplex. Usually, two DNA strands
involved in duplex formation come from two different sources. In case of DNA– RNA duplex, DNA
and RNA may have different origins. Thus, duplex formed are hybrids, and the process of their
formation is hybridization technique. Some of these hybridization techniques are described below.
Southern Blot
It is a technique useful in the detection of specific DNA fragment from mixture of DNA fragments.
A cDNA probe of gene of interest is employed in this technique. This probe hybridizes with the gene
of interest, thus leading to its identification. Different steps involved in southern blot techniques are
given below.
1. The chromosomal DNA is purified and cleaved by restriction endonuclease. Mixtures of DNA
fragments are obtained.
2. The fragments are separated by agarose gel electrophoresis. The gel is prepared from agarose
in a suitable buffer. The DNA fragments are loaded into a well at one end of the gel and
Textbook of Pharmaceutical Biotechnology
210
subjected to electrophoresis. Under conditions of experiment, DNA fragments carry net
negative charge, and hence they move towards anode. Since the gel acts as molecular sieve,
movements of DNA fragments towards anode depends on their sizes. Small fragments move
faster, whereas big fragments get retarded. The DNA fragments are separated according to
their sizes.
3. The DNA in the separated fragments is denatured and made single stranded by soaking gel
first in HCl followed by NaOH solution.
4. The DNA is transferred to nitrocellulose by placing nitrocellulose sheet on the gel. The buffer
in the gel is removed with the help of several blotting papers.
5. The DNA sticks to nitrocellulose, and only the buffer passes through nitrocellulose and
absorbed by a blotting paper. Thus, a perfect nitrocellulose print of the gel is obtained.
6. The nitrocellulose sheet is incubated with buffer containing cDNA probe. cDNA probe is P32
radiolabeled. It hybridizes with gene of interest and excess probe is washed off.
7. Nitrocellulose sheet is dried and placed next to X-ray film. The fragments to which cDNA
probe is bound appears as bands on film when it is developed.
Northern Blot
It is conceptually similar to southern blot. It is mainly used to detect specific RNA fragment from
mixture of RNA fragments.
1. Mixture of RNA fragments are obtained from RNA by action of ribonuclease (RNase).
2. Fragments are separated by agar gel electrophoresis.
3. Nitrocellulose print of fragments is obtained.
Monoclonal cell
Monoclonal antibody
Myeloma cell
PEG
usion
Separation of
Monoclonal cell
ybridoma cell
used cell culture
mmuni ed
Mouse
Antigen
emo al of spleen cell
Figure 13.5 Production of monoclonal antibodies.
Genetic Recombination
211
4. cDNA probe is undertaken and excess probe is washed.
5. Visualization of hybrid on X-Ray film as band.
Western Blot
It is also similar to southern blot in many ways, except in the nature of probe used. In this blotting
technique, radiolabeled ( polyclonal or monoclonal) material is used as probe. This technique is
used to identify a specific protein from mixture of proteins. The steps involved in the technique of
western blot are given below.
1. The protein mixture containing desired protein is subjected to electrophoresis to separate
proteins.
2. The nitrocellulose print is obtained.
3. Antibody probe (radiolabeled) is added.
4. Visualization of antigen– antibody complex is done on X-ray film.
SOME IMPORTANT MEDICINES PRODUCED BY BIOTECHNOLOGY
Humulin
Humulin is the brand name for a group of biosynthetic human insulin products, originally developed
by Genentech in 1978. The generic names are regular insulin, insulin isophane and the Lente series
consisting of Lente (generically known as insulin zinc suspension, a mixture of Ultralente and
Semilente) acquired by Eli Lilly and Company, who arguably facilitated the product’s approval
with the US Food and Drug Administration. The Lente series was discontinued by Eli Lilly and
Company in 2005.
Humulin is synthesized in a laboratory strain of Escherichia coli bacteria that has been
genetically altered with recombinant DNA to produce biosynthetic human insulin. Humulin R
consists of zinc–insulin crystals dissolved in a clear fluid. The synthesized insulin is then combined
with other compounds or types of insulin that affect its shelf life and absorption. For example,
Humulin N is combined with protamine to extend the time-activity profile of Humulin R for an
extended period of time.
1. Humulin R regular U-500 (concentrated) insulin human injection, USP (rDNA origin) is a
stronger concentration (500 units/mL) of Humulin R.
2. Humulin R (regular human insulin injection [rDNA origin]) is short-acting insulin that has a
relatively short duration of activity as compared with other insulins.
3. Humulin 70/30 (70% human insulin isophane suspension, 30% human insulin injection [rDNA
origin]) is a mixture of different types. It is an intermediate-acting insulin combined with the
onset of action of Humulin.
4. Humulin N (human NPH insulin injection [rDNA origin]) is intermediate-acting insulin with
a slower onset of action and a longer duration of activity than Humulin R.
5. Humulin 50/50 (50% human insulin isophane suspension, 50% human insulin injection
[rDNA origin]) is a mixture insulin. It is intermediate-acting insulin combined with the onset
of action of Humulin R.
Textbook of Pharmaceutical Biotechnology
212
Activase
Activase is known as tissue plasminogen activator (tPA), and can be used to treat patients with
acute ischaemic stroke. For certain patients, activase may improve the chances of recovery from
stroke with little or no disability. The patients can receive activase only if they begin treatment
within 3 h after their stroke symptoms start and only after they have had a scan to rule out bleeding
in the brain.
Activase is approved for the treatment of acute myocardial infarction (heart attack) and acute
ischemic stroke (blood clots in the brain) within 3 h of the onset of symptoms. It is also useful in
treatment of acute massive pulmonary embolism (blood clots in the lungs).
Humatrope
Humatrope (somatotropin or somatropin) is a polypeptide hormone of rDNA origin manufactured
by Eli Lilly and Company. It is used to stimulate linear growth in paediatric patients, who lack
adequate normal human growth hormone. It has 191 amino acid residues and a molecular weight
of 22,125 Da.
Its amino acid sequence is identical to that of human growth hormone of pituitary origin
(anterior lobe). Humatrope is synthesized in a strain of E. coli modified by the addition of a gene
for human growth hormone.
Hepatitis B Vaccine ( Recombinant)
Hepatitis B vaccine contains the purified surface antigen (HBsAg) of the hepatitis B virus (HBV).
It is produced by fermenting genetically engineered yeast cells containing a plasmid that carries
the HBsAg. Energix-B (hepatitis B vaccine [ recombinant]) is indicated for immunization against
infection caused by known subtypes of HBV. Hepatitis D (caused by the delta virus) should also
be prevented by Engerix-B, since delta virus replicates only in presence of HBV infection. The
vaccination against HBV has been shown to reduce the overall incidences of HBV infection. It is
also useful in reducing the incidences of the complications of HBV infection, such as chronic active
hepatitis, cirrhosis and primary hepatocellular carcinoma.
Engerix-B does not prevent hepatitis caused by other agents, such as hepatitis A virus,
non-A/non-B hepatitis viruses or other pathogens known to infect the liver.
Vaccination is recommended for persons of all ages or weight and for persons at risk of
exposure to HBV, such as health care personnel, selected patients and patient contacts including
adult haemodialysis patients and patients requiring frequent and/or large volume transfusions of
blood or clotting concentrates. The vaccination is also given to residents and staff of institutions
for the mentally handicapped; subpopulations with a known high incidence of HBV infections,
such as immigrants from Southeast Asia; persons who have travel to areas where HBV infection is
endemic and receive blood from or have close contact with the native populations; infants born to
mothers who are carriers of HBsAg; persons at increased risk of HBV infection due to their sexual
Genetic Recombination
213
practices; prisoners; military personnel identified as being at increased risk, and users of illicit
injectable drugs.
GENE TRANSFER IN HUMANS
The application of gene transfer technology to human gene therapy has been intensively investigated
during the last one decade. Immunotherapies with interleukin-2 and tumour infiltrating lymphocytes
(TILs) have previously been shown to induce regression of metastatic melanoma in approximately
half the patients.
The first experiment in humans was approved by the National Institutes of Health, United
States (January 1989), and permission was granted for the infusion into patients with advanced
melanoma of antilogous TIL that had been subjected to retrovirus-mediated gene transfer.
Techniques of Gene Transfer in Human Cells
Different approaches are considered to deliver a given DNA sequence known as transgene to the
nucleolus of a cell, where this genetic material has to be integrated and potentially be transmitted
to the following generation. One can thereby alter a group of cells in order to produce a protein that
may be directed towards the deficient organ, such as genetically controlled microfactory.
The retroviruses seem to be the most eligible means for efficient gene transfer, but only in cells
undergoing at least one mitotic cycle to obtain integration of the viral genome.
Herpes Virus–Derived Vector
These vectors are defective herpes viruses derived from thermosensitive mutants, incapable of
replicating at temperature above 31°C and processing a natural tropism for the cells of the nervous
system.
Adenoviruses
The recombinant adenoviruses offer first choice alternative to retroviruses when addressing the transfer
of gene into differentiated cells is incapable of proliferating, such as neuron muscular cells, or when the
target is expandable such as pulmonary epithelial cells. Importantly, the recombinant adenoviruses due
to their very high infectious nature, and their ubiquity can be administered in vivo intravenously.
APPLICATIONS OF GENE TRANSFER
Role of Gene Transfer in Human Diseases
The gene transfer has an effective role to play in treatment of different human ailments.
1. Arthritis: The gene therapy has application in a nonlethal disease, rheumatoid arthritis (RA).
Intra-articular transfer of IL-1 receptor antagonist (IL-1Ra) cDNA reduces disease in animal
Textbook of Pharmaceutical Biotechnology
214
models of RA. For establishing safety and feasibility in humans, a phase I clinical study
was carried out involving nine postmenopausal women with advanced RA, who required
unilateral silastic implant arthroplasty of the 2nd–5th metacarpophalangeal (MCP) joints. It
is possible to transfer a potentially therapeutic gene safely to human rheumatoid joints and
to obtain intra-articular transgene expression. This justifies additional efficacy studies and
encourages further development of genetic approaches to the treatment of arthritis and related
disorders.
2. Pulmonary hypertension: The primary pulmonary hypertension associated with progressive
pulmonary hypertension and right
ventricular failure, is a life-threatening disease. The
transfectionof the PGIS gene to the liver resulted in a drug delivery systemof prostacyclin for
the lung, and therefore this may be an effective treatment for pulmonary hypertension. The
intrinsic prostacyclin drug delivery using gene therapy may be useful.
3. Cardiovascular disease: Despite the substantial advances made during the last two decades
in the prevention and treatment of cardiovascular events, cardiovascular disease (CVD) is
still the main cause of deaths in the world. Application of gene therapy in the discipline of
cardiovascular disorders has been the subject of intensive research work during recent times.
The human clinical trials carried out in recent years have shown that the injection of naked
DNA encoding vascular endothelial growth factor promotes collateral vessel development in
patients with critical limb ischaemia or chronic myocardial ischaemia. Application of gene
transfer to other CVDs shall require the development of integrated technologies, as well as a
better definition of cellular and gene targets.
4. Parkinson’s disease: After nearly twenty years of preclinical experimentation with various
gene delivery approaches in animal models of Parkinson’s disease (PD), clinical trials are
finally underway. The risk/benefit ratio for these procedures is now considered acceptable
under approved protocols by regulating agencies. The recombinant adeno-associated viral
vector, a nonpathogenic and non–self-amplifying, is an ideal candidate for gene delivery to
human brain. The candidate genes tested in PD patients encode glutamic acid decarboxylase,
which is injected into the subthalamic nucleus to catalyse biosynthesis of the inhibitory
neurotransmitter aminobutyric acid. It essentially mimics deep brain stimulation of the
nucleus. It also facilitates encoding of aromatic
L-amino acid decarboxylase that converts
L-dopa to dopamine.
5. Cancer: A retroviral vector containing the wild-type p53 gene under control of a `-actin
promoter was produced to mediate transfer of wild-type p53 into human non–small-cell
lung cancers by direct injection. No clinically significant vector-related toxic effects were
observed in nine patients whose conventional treatments failed, for a period of five months
after treatment.
In situ hybridization and DNA polymerase chain reaction demonstrated vector p53
sequences in post treatment biopsies. Apoptosis (programmed cell death) was more frequent
in posttreatment biopsies than in pretreatment biopsies. While tumour regression was observed
in three patients, tumour growth stabilized in other three patients.
6. Growth factor in neuropathic pain: Peripheral nerve injury is a commonly occurring
phenomenon that sometimes results in chronic neuropathic pain with hyperalgesia and
allodynia. The treatment of neuropathic pain is a major clinical challenge because of our poor