ANIMAL BIOTECHNOLOGY

APPLICATION OF rDNA IN ANIMAL CELL

CULTURE

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 Introduction

 Biotechnology- involves the industrial application of living organism

to produce product or process for the betterment of humanity.

 Cell cultures involves the in-vitro cultivation and maintenance of the

cell lines where it generate valuable products based on their genetic
information or due to genes transferred into them using recombinant
DNA technology.

 This has not only resulted in the production of specific biomolecules

in different organism but also has helped to synthesize genetic
material and its related product in the laboratory.

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 Thousands of genes have been cloned and expressed and the
genetic manipulations using r-DNA technology are more
precise and outcomes are more certain over other methods
resulting in faster production of organisms with desired traits.

 In fact, the application of genetic engineering and

recombinant DNA technology has led to the generation of
new classes of organism called Genetically Modified
Organisms (GMO) or Live Modified Organism (LMO),
where manipulations can be made resulting in the products.

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Applications

 Progress in animal cell culture and genetic engineering techniques has made impact in two major areas;

(a) Understanding the biology of the living system by manipulation of genome information
(b) Production of useful metabolites or living organisms having desired metabolic characteristics.

 The most notable applications of the recombinant technology having direct impact on animal cell culture have been:

1. Large scale production of therapeutic protein such as

insulin, hormones,vaccine and interleukins using
recombinant microorganisms.
2. Production of humanized monoclonal antibodies for
therapeutic application.

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<>In Industrial Production of Biomolecules:

 One of the greatest benefit of the

recombinant DNA technology has been
the production of human therapeutics
such as hormones, growth factors and
antibodies which are not only scarcely
available but also are very costly for
human use.
 Ever since the recombinant insulin was
produced by Eli Lilly in 1982,
considerable efforts has been made to
clone and express many therapeutically
important proteins, which are otherwise
difficult to produce either by extraction
from the natural sources or by chemical
synthesis.

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 Types of Biomolecules Produced:

Recombinant Hormones-
Insulin (and its analogs),
Growth hormone,
Follicle stimulating hormone,
Salmon calcitonin.

Blood products-
Albumin,
Thrombolytics,
Fibrinolytics, and
Clotting factors
(Factor VII, Factor IX, tissue plasminogen activator, recombinant hirudin)

Cytokines and growth factors-

Interferon’s,
Interleukins and
Colony stimulating factors
(Interferon, α, β and γ, erythropoietin, interlukin-2, GM-CSF, GCSF)
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 Monoclonal antibodies and related products-
Mouse, chimeric or humanized; whole molecule or fragment; single chain or
bispecific; and conjugated.

Recombinant Vaccines-
Recombinant protein or peptides,
DNA plasmid and
Anti-idiotype (HBsAg vaccine, HPV vaccine)

Recombinant Enzymes-
Dornase– α (Pulomozyme),
Acid glucosidase (Myozyme),
α –L-iduronidase (Aldurazyme) and
Urate Oxidase

Miscellaneous products-
Bone morphogenic protein,
Conjugate antibody,
Pegylated recombinant proteins
(Peg-interferon and peg-antibodies and growth factors)
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Starting with simple protein such as
insulin and then growth hormone,
recombinant biopharmaceuticals has
increased considerably in recent
years.
Till today, around 165
biopharmaceuticals (recombinant
proteins, antibodies, and nucleic acid
based drugs) have been approved
 Therapeutic proteins are preferred over conventional drugs because of-
-- their higher specificity and absence of side effects.
-- less toxic than chemical drugs and are neither carcinogenic
nor teratogenic.

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Recombinant Hormone

INSULIN
• During its first 50 years of use, insulin was extracted from animal sources
(bovine or porcine pancreas). Concerns about purity led to the production of
highly purified, mono component insulin in the 1970s.

• In the 1980s, recombinant-DNA technology and the development of protein-

engineering techniques led to the production of human insulin and modified
insulin analogues with improved pharmacokinetic properties.
Insulin is expressed in recombinant E. coli and then subsequently purified and
refolded in to bioactive form.

• New methods of producing insulin were accompanied by advances in

formulation, which led to the development of rapid-acting insulin’s for use at
meal times and long-acting insulin’s for basal insulin requirements. In
addition, a series of pre-mixed insulin’s that combine the various forms are in
use.

• At present, about 180 individual insulin preparations are available worldwide.

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 Harnessing the Power of Recombinant DNA
Technology – Human Insulin Production by Bacteria

and cut with a restriction enzyme

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join plasmid with the human fragment
Mix the recombinant plasmid with
bacteria.

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 One cell with the
recombinant plasmid

<>This is the step when gene cloning takes place.

<>The single recombinant plasmid replicates within a cell.
<>Then the single cell with many recombinant plasmids produces
trillions of cells with recombinant plasmid and the human insulin gene.

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The final steps are to collect the bacteria, break open the cells,
and purify the insulin protein expressed from the recombinant
human insulin gene.

A fermentor used to grow

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recombinant bacteria.
 • Humulin N [Human
insulin (rDNA origin)
isophane suspension] is a
crystalline suspension of
human insulin with
protamine and zinc
providing an
intermediate-acting
insulin with a slower
onset of action and a
longer duration of activity
HUMULIN® N
(up to 24 hours) than that
of Regular human insulin.

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Overview of gene cloning.

Route to the
Production of
Human Insulin
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 SECOND GENERATION RECOMBINANT
INSULIN
• In normal case of administrating insulin, the plasma
concentration rises slowly and for this reason insulin
injection has to be done at least 15mints before a
meal
decrease in insulin level is also slow, exposing the
patients to a danger of hyperinsulinemia.
All this is due to the existence of therapeutical
insulin as a hexamer (6molecules) which dissociates
slowly to the biologically active dimer /monomer
<> with the help of site directed mutagenesis and
protein engineering second generation recombinant
proteins “muteins” are produced that dissociates
rapidly to biologically active forms

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• INSULIN LISPRO
Modified amino acid residue at position 29 and 30 of B chain and
can be injected immediately before a meal due to its rapid action

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Insulin From E .Coli

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GROWTH HORMONES

hGH
somatotropin
 hUMAN GROWTH HORMONE
• GH are produced from pituitary gland
and stimulate overall body growth by
increasing the cellular uptake of amino
acids and protein synthesis ,
and promote the use of fate as body
fuel

rhGH (recombinant human growth

hormone) : GH
In normal case hGH gene is inserted
into E.Coli plasmid -cultured and hGH
is isolated from the extracellular
medium
-hGH derived from animal cell culture
and is of medical use in case of human
growth deficiency in children and also
in renal carcinoma
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 LIMITATION

During its course of natural production in the body hGH is tagged with a
single peptide (with 26 amino acids )
Single peptide is removed during secretion to release the active hGH for
biological function

In the case of rhGH, signal peptide interrupts the production.

The cDNA synthesized from the mRNA encoding hGH is integrated in to the
plasmid and then inserted in to E.Coli -cultured and hGH along with signal
peptide is isolated.
-E.Coli cannot remove the signal peptide and is very difficult to remove by
any other means. [no Restriction Endonuclease available]

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NOVEL APPROACH

<>Resolved the problem of signal peptide interruption.

The base sequence in cDNA encoding signal peptide (26 amino acids) plus
the neighbouring 24 amino acids (total 50 amino acids ) is cut by EcoR1 RE
This deleted 24 amino acid sequence of hGH is freshly prepared and ligated
to the remaining hGH cDNA
- then integrated with a plasmid
- inserted in to E.Coli
- cultured to release hGH [with out any signal peptide]

<>Production of rhGH in CHO cells {Chinese hamster ovary cells} is an

alternative to production in Escherichia coli,
- with the advantage that rhGH is secreted into protein-free production media,
facilitating a more simple purification & avoiding resolubilization of
inclusion bodies and protein refolding.

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 recombinant hGH
<>PROTROPIN -Genetech company
<>HUMATROPE – Eri Lilly company

PROTROPIN

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 Somatotropin:
Growth hormone, a polypeptide containing 191 amino acids,
produced by the anterior pituitary, the front section of the pituitary
gland. It acts by stimulating the release of another hormone called
somatomedin by the liver, thereby causing growth.

 It refers to the growth hormone produced natively and naturally in

animals, whereas the termSomatropin refers to growth hormone
produced by recombinant DNA technology and is abbreviated
"rhGH" in humans.

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• Recombinant DNA technology has
permitted the expression of
heterologous protein in host cells such
as E. coli bacteria.

• In the case of the growth hormone

somatotropin, the protein is sequestered
in refractile bodies within the
cytoplasm of the host cells.

• The refractile bodies may be recovered

from the host cell culture by disrupting
the cell so as to release the refractile
bodies, and thereupon collecting the
refractile bodies as a solid pellet by
differential centrifugation.

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• The refractile bodies are solubilized in an aqueous solution of a suitable
chaotropic agent such as urea or guanidine hydrochloride at an alkaline pH (9-
12).

• The solubilized proteins are subsequently naturized by contact with a mild

oxidizing agent to form intramolecular disulfide bonds while refolding the
protein to its biologically active native conformation.

• Biologically active somatotropins are effective to enhance animal growth and

productivity when administered parenterally as by subcutaneous or
intramuscular injection or implantation.

• Bovine somatotropin [bST] for example, is effective to increase milk

production of lactating cows, while porcine somatotropin is effective to
improve feed efficiency and lean to fat ratio when administered to finishing
hogs.

• Somatotropin is also being used in chronic renal insufficiency and Turner’s

syndrome
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 bST:
 is a GH produced in cattle by the pituitary gland located at the base of the
animals brain.

 In the 1930s, it was discovered that injecting bST into lactating (milk-
producing) cows significantly increased milk production.
But the only source of bST was from the pituitary glands of slaughtered
cattle. There were only small quantities of bST available, and it was very
expensive.

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 Now with the aid of rDNA, scientists have determined the gene that controls
or codes for the production of bST. They have removed this gene from cattle
and inserted it into a bacterium called Escherichia coli

 E.Coli acts like a tiny factory and produces large amounts of bST in
controlled laboratory conditions. The bST produced by the bacteria is
purified and then injected into cattle

 To affect a cows milk production, bST must be injected into the animal on a
regular basis, similar to the way insulin must be regularly injected into
people who have certain types of diabetes

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 Erythropoietin:
It’s a hormone synthesized by the kidneys and stimulate the stem cells of
bone marrow to produce mature erythrocytes.
Erythropoietin ‘a’ and ‘b’ derived from animal cell culture and are of
significant use as therapeutic agents.
The former is used for the treatment of anaemia resulting from cancer and
chemotherapy and the latter to treat anaemia secondary to kidney disease.

Recombinant Erythropoietin
<> EPOGEN - treatment of anemia
<> PROCRIT- act like natural hormone and stimulate the
production of erythrocytes
- an alternative to blood transfusion
[But very Expensive]
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CLOTTING FACTOR V111
 Factor VIII (FVIII) is an essential
blood clotting factor also known as anti-
hemophilic factor (AHF). In humans, Factor
VIII is encoded by the F8 gene.
Defects in this gene results in hemophilia A a
well known recessive
X-linked coagulation disorder (prolonged
clotting time resulting in internal bleeding)

FVIII is a glycoprotein procofactor.

It has been found to be synthesized and
released into the bloodstream by the vascular,
glomerular, and tubular endothelium, and the
sinusoidal cells of the liver

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 Recombinant Factor [F8]
Gene for the formation of factor V111 is located in X chromosome
- complex gene 186kb in size ,organized in to 26 Exons of varying
length
- in between Exons many Introns are also present

With the aid of rDNA technology mature mRNA [only EXONS]

responsible for the synthesis of F8 is isolated
- cDNA are synthesized for mature mRNA
- inserted into mammalian cells / hamster kidney
- recombinant F8 is isolated

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rFactor 8

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 tPA
TISSUE PLASMINOGEN ACTIVATOR
• tPA is a naturally occurring protease enzyme that helps to dissolve blood
clots

tPA
PLASMINOGEN PLASMIN

FIBRINOGEN FIBRIN
BLOOD CLOT

Degraded Soluble Products

In normal case PLASMIN degrade FIBRIN and dissolve blood clots.

This plasmin is actually produced by activation of plasminogen by tPA

<> tPA is of high therapeutical value –removing

arterial thrombi {blood clot}, the possible damage
caused by them on heart and brain.
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 Recombinant tPA:
- cDNA is synthesized for tPA
- cDNA then attached to a synthetic plasmid and introduce to mammalian
cells.
- Cells cultured and tPA producing cells are then transferred to an industrial
tank [fermenter]
- tPA secreted in to the culture medium is isolated

<> tPA is the 1st pharmaceutical product to be produced by mammalian cell

culture
Eg; ACTIVASE
2nd generation tPA -AITEPLASE and RETEPLASE

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Ab-PA Conjugates:

- antibody against fibrin [antifibrin antibody] can be conjugated with

tissue plasminogen activator
[ immunotherapeutic thrombolytic agent ]

- it quickly and specifically binds to fibrin clots and locally increase the
conversion of plasminogen to plasmin to dissolve fibrin

tPA
Conjugate fibrin clots degradation
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 ADVANTAGE:
<>tPA acts on blood clots and degrade with out reducing the blood clotting
capability elsewhere.

<>can be administered intravenously [urokinase & streptokinase - need to be

administered directly to the blocked blood vessel ]

<>action much faster than other thrombolytic agents

<>reduced side effects

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INTERFERONS
 Interferon’s are proteins produced by a cell infected by a virus and provide
protection to other healthy cells from viruses.
It is defined as

“a protein which exerts virus non-specific antiviral activity, atleast in

homologous cells through cellular metabolic procedures involving the
synthesis of both RNA and protein”.

Thus, interferon is secreted by human cells just to resist the immediate

invasion by virus and multiplication of abnormal cells.

 Interferon is used to cure many viral diseases such as common cold and
hepatitis. Recombinant techniques have made the production of biologically
modified interferons with enhanced specific activity-Human Interferon
Genes {HIG}.

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Overview On action of IFN.

IFN

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Recombinant IFN
- cDNA was synthesized from the mRNA of a specific IFN
- Integrated with a plasmid vector
- Introduced to a E.Coli cell
- IFN isolated from culture medium

 Mouse fibroblast cultures as well as human leukocyte cultures in vitro are used
as the sources of interferon production.
Production of IFN is relatively less in bacterial hosts ,mainly because most IFN
are glycoproteins in nature and bacteria do not possess the machinery for
glycosylation
• IFN from Yeast-
Yeast is the most suitable vector for the production of rIFN as they possess
the mechanism to carry out glycosylation of protein

- DNA sequence coding for specific human IFN is isolated

- attached to yeast alcohol dehydrogenase gene in a plasmid
- introduced to yeast cells
- recombinant human IFN are obtained

HYBRID IFN: different IFN with different antiviral activities combined to

produce HYBRID IFN

APPLICATION:
- treatment of large number of viral diseases and cancer
- also in the treatment of common colds and influenza [nasal sprays]

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IFN-β
1 Human Fibroblast

2 Human IFN-β gene

3 Mdified Human
IFN-β

4 Incorporated in to
plasmid

5 Inserted in to E.Coli

6 E.Coli cells
replicate and
produce beta IFN

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VACCINES
 Vaccines are chemically substance prepared from the proteins (antigen) of
other animals which offer immunity to a particular virus.

 Many different types of vaccines are biologically synthesized through

genetic engineering {rDNA technology}.
-Production of FMD vaccines Is the most important example of the use of
large scale cell cultures.

 There are several other vaccines including polio vaccine, bovine leukemia
virus (BLV) vaccines, rabies vaccines, etc., which are produced at
commercial scale using cell cultures.

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 Eg;

Recombinant Vaccines for Hepatitis B Virus:

 [HBV] were produced by cloning HBsAg gene of the virus in yeast cells.

 The yeast system has its complex membrane and ability of secreting
glycosylate protein. This has made it possible to build an autonomously
replicating plasmid containing HBsAg gene near the yeast alcohol
dehydrogenase (ADH) promoter. [pMA 56 yeast vector]

 Recombinant plasmid is then inserted in to yeast cell and multiplied in

trytophan-free medium.

 The transformed cells are selected and its high immunogenicity has
made it possible to market the recombinant product as vaccine against
HBV infection.

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[HBV] production:

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 Vaccines derived from rDNA technology are purer,
safer and more efficacious and are already being
used for clinical trials.

In order to produce killed virus vaccine, the

vaccine is usually subjected to a concentration step
after virus inactivation. The loss of potency is
prevented by adding suitable stabilizers.

 The vaccines are then stored at low temperatures

till use. The entire operation must be carried out
under strict quality control.

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MONOCLONAL ANTIBODIES
 Monoclonal antibodies (mAb) are important reagents used in biomedical
research, in diagnosis of diseases, and in treatment of such diseases as
infections and cancer.

 These antibodies are produced by cell lines or clones obtained from

animals that have been immunized with the substance that is the subject of
study.

 To produce the desired mAb, the cells must be grown in either of two
ways:
by injection into the abdominal cavity of a suitably prepared mouse
or by tissue culturing cells in plastic flasks.
Further processing of the mouse ascitic fluid and of the tissue culture
supernatant might be required to obtain mAb with the required purity and
concentration. The mouse method is generally familiar, well understood,
and widely available in many laboratories.
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 prod
uctio
m Ab n

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 The use of monoclonal antibodies (mAb) in biomedical research with the
aid of rDNA technology has been and will continue to be important for the
identification of proteins, carbohydrates, and nucleic acids.

 These advances is basic biologic sciences have improved our

understanding of the host response to infectious-disease agents and toxins
produced by these agents, to transplanted organs and tissues, to
spontaneously transformed cells (tumors), and to endogenous antigens
(involved in autoimmunity).

 Despite all those benefits associated with production of mAb with the
mouse ascites method, it can be distressful to the host animal.

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TRANSGENIC ANIMALS
 A transgenic animal is one that carries a foreign gene that has been
deliberately inserted into its genome. The foreign gene is constructed using
recombinant DNA methodology.
<>Transgenic sheep and goats have been produced that express foreign
proteins in their milk.
<>Transgenic chickens are now able to synthesize human proteins in the
"white" of the eggs.

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Transgenic Mice:
 Transgenic mice have provided the tools for exploring many biological
questions. Normal mice cannot be infected with polio virus. They lack the cell-
surface molecule that, in humans, serves as the receptor for the virus. So
normal mice cannot serve as an inexpensive, easily-manipulated model for
studying the disease. However, transgenic mice expressing the human gene for
the polio virus receptor
-can be infected by polio virus and even
-develop paralysis and other pathological changes characteristic of the disease
in humans.

 The Embryonic Stem Cell and Pronucleus Method are the commonly
employed recombinant method for producing transgenic mice

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Transgenic Mice:
Transgenic Sheep:
 The rate of transgenesis in sheep is very low (0.1 to 0.2%). This can be
improved by recombinant technique in which transgenic viable embryos
are transferred to surrogate ewes (female sheep).

 Although microinjection is the most common method for DNA delivery,

gene targeting may be increasingly used in future.

 In this approach, embryonic stem (ES) cells in culture arc transfected with
a vector which targets the gene to a particular site by homologous
recombination. This technique, though successfully used in mice, has yet
to be applied to sheep, where ES cells will have to be isolated first.

 These animals should eventually prove to be valuable sources of proteins

for human therapy.
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AAT Protein production from transgenic sheep

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 Thus,recombinant DNA technology has been the major
backbone of modern biotechnology especially in the field of
animal cell culture that has made the today’s science for
mankind

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Thank U…..