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 HormonesInsulin (and its analogs), Growth hormone, Follicle stimulating hormone, Salmon calcitonin. Blood productsAlbumin, Thrombolytics, Fibrinolytics, and Clotting factors (Factor VII, Factor IX, tissue plasminogen activator, recombinant hirudin) Cytokines and growth factorsInterferon’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 productsMouse, chimeric or humanized; whole molecule or fragment; single chain or bispecific; and conjugated. Recombinant VaccinesRecombinant protein or peptides, DNA plasmid and Anti-idiotype (HBsAg vaccine, HPV vaccine) Recombinant EnzymesDornase– α (Pulomozyme), Acid glucosidase (Myozyme), α –L-iduronidase (Aldurazyme) and Urate Oxidase Miscellaneous productsBone 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 proteinengineering 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. ..Mj

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 recombinant bacteria.

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• 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 (up to 24 hours) than that of Regular human insulin.

HUMULIN® N

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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 ..Mj

• 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) : 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

GH

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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 (912). • 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 (milkproducing) 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 antihemophilic 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 ..Mj

rFactor 8

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tPA TISSUE PLASMINOGEN ACTIVATOR

tPA is a naturally occurring protease enzyme that helps to dissolve blood clots PLASMINOGEN tPA PLASMIN FIBRIN BLOOD CLOT Soluble Products

FIBRINOGEN

Degraded

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. ..Mj

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 ..Mj

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 YeastYeast 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] ..Mj

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:
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[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. ..Mj

prod uctio n m Ab

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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 cellsurface 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. ..Mj

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…..