Sie sind auf Seite 1von 45

http://www.molecular-plant-biotechnology.info/recombinant-DNA-technology/steps-in-genecloning.

htm

Recombinant DNA Technology - In nature, gene transfers are rather imprecise, and their range, in tenns of species involved, is remarkably limited. The above problems are circumvented by the recombinant DNA technology. A recombinant DNA molecule is produced by joining together two or more DNA segments usually originating from different organisms. More specifically, a recombinant DNA molecule is a vector into which the desired DNA fragment has been inserted to enable its cloning in an appropriate host. This is achieved by using specific enzymes for cutting the DNA (restriction enzymes) into suitable fragments and then for joining together the appropriate fragments (ligation). In this manner, a gene may be produced, which contains the. coding region from one organism joined to regulatory sequences from another organism; such a gene is called chimaeric gene. Clearly, the capability to produce recombinant DNA molecules has given man the power and opportunity to create novel gene functions to suit specific needs. Recombinant DNA molecules are produced with one of the following three objectives: (1) to obtain a large number of copies of specific DNA fragments, (2) to recover large quantities of the protein produced by the concerned gene, or (3) to integrate the gene in question into the chromosome of a target organism where it expresses itself. Even for the latter two objectives, it is essential to first obtain a large number of copies of the concerned genes. To achieve this, the DNA segments are integrated into a self-replicating DNA molecule called vector; most commonly used vectors are either bacterial plasmids or DNA viruses. All these steps concerned with piecing together DNA segments of diverse origin and placing them into a suitable vector together constitute recombinant DNA technology. The DNA segment to be cloned is called DNA insert. Recombinant DNAs are introduced into a suitable organism, usually a bacterium; this organism is called host, while the process is called transformation. The transformed host cells are selected and cloned. The recombinant DNA present in such clones would replicate either in synchrony with or independent of the host cell; the gene present in 'the vector mayor may not express itself, i.e., direct the synthesis of concerned polypeptide. The step concerned with transformation of a

suitable host with recombinant DNA, and cloning of the transformed cells is called DNA cloning or gene cloning. However, often DNA or gene cloning is taken to include both the development of recombinant DNAs as well as their cloning in a suitable host. Similarly, often the term recombinant DNA technology is used as a synonym for DNA or gene cloning used in the broader sense. A rather popular term for these activities is genetic engineering. A clone consists of asexual progeny of a single individual or cell, while the process/technique of producing a clone is called cloning. As a result, all the individuals of a clone have the same genotype, which is also identical with that of the individual from which the clone was derived. Therefore, the genomes present in members of a single clone are also identical; this applies to the recombinant DNA as well. Therefore, gene or DNA cloning produces large numbers of copies of the gene/DNA being cloned.

Restriction Endonucleases - Endonucleases are enzymes that produce internal cuts, called cleavage, in DNA molecules. Many endonucleases cleave DNA molecules at random sites. But a class of endonucleases cleaves DNA only within or near those sites, which have specific base sequences; such endonucleases are known as restriction endonucleases, and the sites recognised by, them are called recognition sequences or recognition sites. The recognition sequences are different and specific for the different restriction endonucleases or restriction enzymes.Restriction enzymes were discovered due to and named after the phenomenon of host restriction of bacterial phages. The presence bf restriction enzymes was postulated by W. Arber during 1960s, while the first true restriction endonuclease was isolated in 1970. Smith, Nathans and Arber were awarded the Nobel Prize for Physiology and Medicine in 1978 for the discovery of endonucleases. Restriction endonucleases are indispensable for DNA cloning and sequencing. They serve as the tools for cutting DNA molecules at predetermined sites, which is the basic requirement for gene cloning or recombinant DNA technology.

Restriction modification system


From Wikipedia, the free encyclopedia

The restriction modification system (RM system) is used by bacteria, and perhaps other prokaryotic organisms to protect themselves from foreign DNA, such as the one borne by bacteriophages. This phenomenon was first noticed in the 1950s. Certain bacteria strains were found to inhibit (restrict) the growth of viruses grown in previous strains. This effect was attributed to sequence-specific restriction enzymes.

Bacteria have restriction enzymes, also called restriction endonucleases, which cleave double stranded DNA at specific points into fragments, which are then degraded further by other endonucleases. This prevents infection by effectively destroying the foreign DNA introduced by an infectious agent (such as a bacteriophage). Approximately one quarter of known bacteria possess RM systems and of those about one half have more than one type of system. Restriction enzymes only cleave at specific sequences of DNA which are usually 4-6 base pairs long, and often palindromic. Given that the sequences that the restriction enzymes recognize are very short, the bacterium itself will almost certainly have many of these sequences present in its own DNA. Therefore, in order to prevent destruction of its own DNA by the restriction enzymes, the bacterium marks its own DNA by adding methyl groups to it. This modification must not interfere with the DNA base-pairing, and therefore, usually only a few specific bases are modified on each strand.

Contents
[hide]

1 Types of restriction modification system 2 Uses 3 See also 4 References

[edit] Types of restriction modification system


There are Four kinds of restriction modification system: type I, type II, type IIS, type III and Type IV, all with restriction enzyme activity and a methylase activity. They were named in the order of discovery, although the type II system is the most common. Type I systems are the most complex, consisting of three polypeptides: R (restriction), M (modification), and S (specificity). The resulting complex can both cleave and methylate DNA. Both reactions require ATP, and cleavage often occurs a considerable distance from the recognition site. The S subunit determines the specificity of both restriction and methylation. Cleavage occurs at variable distances from the recognition sequence, so discrete bands are not easily visualized by gel electrophoresis. Type II systems are the simplest and the most prevalent. Instead of working as a complex, the methyltransferase and endonuclease are encoded as two separate proteins and act independently (there is no specificity protein). Both proteins recognize the same recognition site, and therefore compete for activity. The methyltransferase acts as a monomer, methylating the duplex one strand at a time. The endonuclease acts as a homodimer, which facilitates the cleavage of both strands. Cleavage occurs at a defined position close to or within the recognition sequence, thus producing discrete fragments during gel electrophoresis. For this reason, Type II systems are used in labs for DNA analysis and gene cloning.

Type III systems have R and M proteins that form a complex of modification and cleavage. The M protein, however, can methylate on its own. Methylation also only occurs on one strand of the DNA unlike most other known mechanisms. The heterodimer formed by the R and M proteins competes with itself by modifying and restricting the same reaction. This results in incomplete digestion.[1][2]

[edit] Uses
RM systems can be cloned into plasmids and selected because of the resistance provided by the methylation enzyme. Once the plasmid begins to replicate, the methylation enzyme will be produced and methylate the plasmid DNA, protecting it from a specific restriction enzyme. Some viruses have evolved ways of subverting the restriction modification system, usually by modifying their own DNA, by adding methyl or glycosyl groups to it, thus blocking the restriction enzymes. Other viruses, such as bacteriophages T3 and T7, encode proteins that inhibit the restriction enzymes. To counteract these viruses, some bacteria have evolved restriction systems which only recognize and cleave modified DNA, but do not act upon the host's unmodified DNA. Some prokaryotes have developed multiple types of restriction modification systems.

Types of Restriction Endonucleases - There are three distinct types of restriction endonucleases. Type I restriction endonucleases are complex endonucleases, and have recognition sequences of about 15 bp; they cleave the DNA about 1000 bp away from the 5'-end of the sequence "TCA" located within the recognition site, e.g., EcoK, EcoB etc. Type II restriction endonucleases are remarkably stable and induce cleavage either, in most cases, within their recognition sequences or very close to them. More than 350 different type II endonucleases with over 100 different recognition sequences are known. They require Mg2+ ions for cleavage. The first type II enzyme to be isolated was Hind II in 1970. Only type II restriction endonucleases are used for restriction mapping and gene cloning. Type III restriction endonucleases are intermediate between the type I and type II enzymes; they cleave DNA in the immediate vicinity of their recognition sites, e.g., EcoPl, EcoP15, Hind III etc. Nomenclature of Restriction Endonucleases - The nomenclature of restriction endonucleases follows a general pattern. (1) The first letter of the name of genus in which a given enzyme is first discovered is written in capital. (2) This is followed by the first two letters of species name of the organism. These three letters

are generally written in italics, e.g., Eco from Escherichia coli, Hin from Haemophilus influenzae, etc. (3) Strain or type identification is depicted next in Roman. e.g., Ecok; (4) When an organism produces more than one enzyme, they are identified by sequential Roman numerals, e.g., the different enzymes produced by H. influenzae strain Rd are named Hind II, Hind III, etc. ome Restriction Endonucleases Restriction endonuclease AvaI* AluI BamHI EcoRI EcoRII** BglII HindII HindIII HindII HpaI HpaII HinfI* NIaIII PstI Sau3A TaqI Source (organism an strain) Anabaena variabilis (A TCC 27892) Arthrobacter luteus Bacillus amyloliquefaciens H Escherichia coli Ry13 E. coli R245 Bacillus globigli Haemophilus influenzae Rd H. influenzae Rd H. influenzae PI H. parainfluenzae H. parainfluenzae H. influenzae Rf Neisseria lactamica Providencia stuartii Staphylococcus aureus 3A Thermus aquaticus YTI Recognition sequence C/Py CG Pu G G Pu GC Py/C AG/CT TC/GA G/G AT CC C C TA G/G G/AA TT C C TT AA/G /CCA (T) GG GGT(A)CC/ A/G ATCT TC TAG/A GT Py/Pu AC CA Pu/Py TG A/A GC T T T T CG A/A G/G C C C C G/G GTT/ AAC CAA/TTG C/C GG GG C/C G/A NT C C T NA/G CATG/ /GTAC C T GCA/G G/A CGTC /GATC CT AG/ T/C GA AG C/T

* Pu, either of the two purines (adenine or guanine) Py, either of the two pyrimidines (thymine or cytosine) N, any of the four bases (adenine, guanine, thymine or cytosine) ** The base given within parenthesis, e.g., (T) may occur in place of the preceding base, e.g., A. Recognition Sequences For Type II Endonucleases - The recognition sequences for Type II endonucleases form palindromes with rotational symmetry. In a palindrome, the base sequence in the second half of a DNA strand is the mirror image of the sequence in its first half; consequently, the complementary DNA strand of a double helix also shows the same situation. But in a palindrome with rotational symmetry, the base sequence in the first half of one strand of a DNA double helix is the mirror image of the second half of its complementary strand. Thus in such palindromes, the base sequence in both the strands of a DNA duplex reads the same when read from the same end (either 5' or 3') of both the strands. Most of the type II restriction endonucleases have recognition sites of 4, 5 or 6 bp (base pairs), which are predominantly GC-rich. Longer palindromic target sequences are also known, and so are nonpalindromic ones (specific for some enzymes). Some restriction enzymes have ambiguities in their recognition sites, e.g. EcoRII, so that they may recognise upto 4 different target sequences. Cleavage Pattern of Type II Restriction Endonucleases - Most type II restriction endonucleases cleave the DNA molecules within their specific recognition sequences, but some produce cuts immediately outside the target sequence, e.g., NlaIII, Sau3A, etc. These cuts are either (1) staggered or (2) even, depending on the enzyme. Most enzymes produce staggered cuts in which the two strands of a DNA double helix are cleaved at different locations; this generates protruding (3'- or 5'-) ends, i.e., one strand of the double helix extends some bases beyond the other: Due to the palindromic (symmetrical) nature of the target sites, the two protruding ends generated by such a cleavage by a given enzyme have complementary base sequence. As a result, they readily pair with each other; such ends are called cohesive or sticky ends. An important consequence of this fact is that when fragments generated by a single restriction enzyme from different DNAs are mixed, they join together due to their sticky ends. Therefore, this property of the restriction enzymes is of great value for the construction of recombinant DNAs. Some restriction enzymes, on the other hand, cut both the strands of a DNA molecule at the same site so that the resulting termini or ends have blunt or flush ends in which the two strands end at the same point. The blunt cut ends also can be effectively utilized for construction of recombinant DNAs following one of several strategies.

Modification of Cut Ends - The 3'-ends of DNA strands always carry a free hydroxyl (-OH) group, while their 5'-ends always bear a phosphate group. Often the ends produced by restriction enzymes have to be modified for further manipulation of the fragments; some of the modifications are summarised below. 1. Removal of the 5'-phosphate group of vector DNA by alkaline phosphatase treatment in order to prevent vector circularization during DNA insert integration. 2. Addition of a phosphate group to a free 5'-hydroxyl group by T4 polynucleotide kinase. 3. Removal of the protruding ends by digestion with, say, S1 nuclease; this enzyme digests both 3'- and 5'-protruding ends. 4. Filling in of the protruding ends by extending the recessed (shorter) strand with, say, Klenow fragment of E. coli DNA polymerase I. (Both the strategies 3 and 4 generate blunt ends which can be ligated by T4 polynucleotide ligase.) 5. Synthesis of single-stranded tails (protruding ends) at the 3'-ends of blunt ended fragments by the enzyme terminal deoxynucleotidyl transferase; this is called tailing. This reaction can be used to generate protruding ends of defined sequence, e.g., poly-A tails on the 3'ends of the DNA insert and poly- T tails on the 3'-ends of the vector; the protruding ends of the DNA insert and the vector will, therefore, base pair under annealing conditions. 6. Linker and/or adaptor molecules can be joined to the cut ends. Linkers are short, chemically synthesized, self complementary, double stranded oligonucleotides, which contain within them one or more restriction endonuclease sites, e.g., linker 5' -CCGAA TTCGG (only one strand of the linker is shown here) contains one EcoRI site. Linkers are joined with blunt ended DNA fragments; cleavage of the linker with the appropriate restriction enzyme creates suitable cohesive protruding ends Linkers create cohesive ends Oft blunt ended DNA fragments, and on fragments having unmatched or undefined sequences in their protruding ends. In the latter situation, the DNA fragments are first made blunt-ended, following which the selected linkers are ligated to them by T 4 ligase. 7. Adaptors are short, chemically synthesized- DNA double strands, which can be used to link the ends of two DNA molecules that have different sequences at their ends. There are different kinds of adaptors suited for different purposes. For example, a conversion adaptor is used to join a DNA fragment or insert cut with one restriction enzyme, say, EcoRI, with a vector opened with another enzyme, e.g., BamHI. These adaptors have the recognition sequences of different endonucleases at their ends. For example, the conversion adaptor has recognition sequence for BamHI at one end and that for EcoRI at the other. This adaptor can be used to convert the cohesive end generated by BamH1 to one produced by EcoRI or vice versa.

Steps in Gene Cloning - The entire procedure of cloning or recombinant DNA technology may be classified into the following five steps for the convenience in description and on the basis of the chief activity performed. 1. Identification and isolation of the desired gene or DNA fragment to be cloned. 2. Insertion of the isolated gene in a suitable vector. 3. Introduction of this vector into a suitable organism/cell called host (transformation). 4. Selection of the transformed host cells. 5. Multiplication/expression/integration followed by expression of the introduced gene in the host.

Selection of Recombinant Clones - When recombinant DNA is constructed and used for transformation of E. coli, cells following types of bacterial cells are obtained: (1) majority of the cells are nontransformed, (2) a proportion of the transformed cells contain unaltered vector, while (3) the remainder cells have recombinant DNA. The first objective of cloning experiments is to identify and isolate those small number of cells that contain the recombinant DNA from among a very large number of nontransformed cells. Since the DNA inserts are generally mixtures, particularly when cDNA preparations and genomic DNA fragments are used, the various transformed clones would contain a variety of different DNA inserts. The next step, therefore, is to identify the clone having the desired DNA insert from among the large number of clones containing the recombinant DNAs. Suitable selection strategies have been devised to achieve these two critical objectives; this is the most important step in DNA cloning. Identification of Clones Having Recombinant DNAs - The second step consists of identification and isolation of those clones that are transformed by the recombinant DNAs from among those that contain the unaltered vector. This may be achieved in one of several ways listed below. 1. In case the vector has two selectable markers, e.g., pBR322, the DNA insert may be placed within one of these markers, say, ampT gene. The other marker, in this case, tetr, is used for

elimination of the nontransformed cells. The transformed clones are then replicaplated on ampicillin containing medium. The clones containing the recombinant DNAs will be sensitive to ampicillin due to inactivation of the gene ampT by insertion of the DNA fragment. Such clones are identified and isolated from the master plate. 2. Some vectors contain a gene, or sometimes only part of a gene, which complements a function missing in their host cells, e.g., gene lacZ in the pUC vectors, which complements such lacZ- E. coli strains in which lacZ is deleted. The same combination is used for some A. vectors and M13 phage vectors. In all such cases, the DNA insert is so placed that it disrupts the expression of lacZ. Therefore, E. coli cells containing the recombinant DNA are deficient in -galactosidase and produce white colonies or plaques on a medium containing X-gal and IPTG. On the other hand, tells having the unchanged vector produce active -galactosidase and give rise to blue colonies or plaques on the same medium. This allows an easy identification of the clones containing the recombinant DNAs. 3. When the DNA insert codes for a gene product, which is defective in the auxotrophic host cells, a direct selection for the recombinant DNA is possible. The host cells are grown on a medium lacking the compound needed by the auxotrophic host; only those cells, which contain the recombinant DNA can grow and form colonies. Obviously, this approach is limited in application. 4. Similarly, selection by suppression of nonsense mutations present in the host also permits a direct selection for the recombinant DNA. 5. Some A. vectors retain the lysogenic function as well, e.g., gt10. In such vectors, the DNA insert may be placed within the lysis repressor gene cI- so that the vector becomes cI. As a result, cells transfected by the recombinant DNA will give rise to clear plaques, whereas those infected by the unaltered vector will yield cloudy or turbid plaques. Thus the recombinant DNAs are readily identified and isolated. 6. Some vectors, e.g., A. replacement vectors and cosmids, are much shorter than the minimum genome length needed for their packaging within virus particles. In such cases, the length of DNA insert can be so adjusted as to allow the packaging of only the recombinant DNA. This provides an efficient selection strategy for recombinant DNA. Selection of Clone Containing A Specific DNA Insert -Once we obtain a population of recombinant clones the next step is to identify a clone, which has the DNA insert of interest. The technique used for identification has to be highly precise and extremely sensitive to allow an accurate detection of a single clone from among the thousands obtained from a cloning experiment. The various strategies used for the purpose are briefly outlined below.

Colony Hybridization. The most efficient and rapid strategy for identification of a clone having the desired insert uses the technique of colony hybridization. The bacterial colonies are replicaplated or phage plaques are directly lifted on nitrocellulose filters, the cells are lysed and their DNA is denatured, the filter is incubated with the specific radioactive 32p-labelled) probe under anealing conditions. After some time, the probe is washed out leaving only those probe molecules that have hybridized with the denatured DNA from bacterial cells or phage particles. The colonies/plaques with whose DNA the probe has hybridized are identified by autoradiography; these contain the desired DNA insert. These colonies/plaques are isolated from the master plate used for replica plating.A very large number of colonies or plaques (upto 10,000 plaques) can be lifted on to a single 10 cm diameter filter. But it is essential that a specific probe for the DNA insert is available. A probe is a polynucleotide (DNA or RNA; usually small molecules of as few as 15 bases, but more often of 2530 bases) molecule of a specific base sequence, which is used to detect DNA molecules having the same base sequence by complementary base pairing. Generally, the probes are labelled with 32p to enable autoradiography for an easy identification of the DNA samples that base-pair with the probe. It is desirable that the probes are single-stranded to avoid pairing between the two strands of the probe itself. Either DNA or RNA can be used as probe. There are several approaches for developing specific probes. Other Approaches. When specific probes are not available, many indirect approaches may be used for the identification of clones having the desired DNA insert. These procedures are not generally convenient for screening of a large number of clones. Two of such procedures, called (1) hybrid arrested translation (HART) and (2) hybrid selection, use in vitro translation systems and then identification of the resulting polypeptide(s).It is, therefore, necessary that the protein product of the DNA insert being searched should be known, at least in terms of its electrophoretic mobility. Complementation. The cloned DNA insert may express itself in the bacterial cells; this is possible for prokaryotic genes, some yeast genes and for eukaryotic cDNAs cloned in suitable expression vectors. Eukaryotic sequences isolated from genomic DNA have to be expressed in appropriate eukaryotic hosts, e.g., yeast cells, animal cells m culture, etc. If the protein produced by the desired DNA insert is deficient in the

host cells, this insert will correct the deficiency of the cells transformed by it, i.e., will complement the deficiency of host cells. This can be stated in general terms as follows. The host cells are deficient in a protein A, i.e., they are A-. These cells can be used to isolate the DNA fragment coding for protein A from a mixture of DNA fragments. Expression of recombinant DNAs are prepared from the DNA fragments and A- host cells are transformed; these cells are now cultured under selective conditions that require functional A product. Only those host cells that contain the DNA insert encoding protein A will be able to multiply under the selective conditions (since the DNA insert will provide functional protein A). This strategy is limited in

application by the availability of appropriate host cells. Unique Gene Products. Alternatively, the protein product of DNA insert can be identified by its unique function, i.e., a function not performed by the proteins of nontransformed host cells. Such functions may relate to enzyme activities or hormone effects for which appropriate assays exist. Antibodies Specific to the Protein Product. Finally, if the protein lacks a recognizable and measurable function, it can be detected by using specific antibodies. A practical approach is to divide the large number of recombinant clones into a convenient number of groups and to assay for the presence of the protein. The positive group is again divided into subgroups and assayed. In this manner, the positive groups are subdivided again and again till a single positive clone is identified. This approach is applicable to the previous strategy as well. The identification of proteins using antibodies may be achieved by western blotting, precipitation and electrophoresis or ELISA (enzyme-linked immunosorbent assay; Appendix-2.IX). Colony/Plaque Screening with Antibodies. An efficient and rapid screening using antibodies is as follows. The antibody specific to the concerned gene product (i.e., protein) is spread uniformly over a solid support, e.g., plastic or paper disc, which is placed in contact with an agar layer containing lysed bacterial colonies or phage plaques. If any clone is producing the protein in question, it will bind to the antibody molecules present on the disc. The disc is removed from the agar, is treated with a second radiolabelled (generally with L25I) antibody, which is also specific to the same protein but in a region different from that recognised by the first antibody. These antibodies, therefore, will also bind to the protein moleculed held by the first antibody; the location of radioactivity on the disc is determined by autoradiography. The colonies/plaques producing the protein are then identified and isolated from the master plate. This technique is analogous to colony hybridization and is able to screen large numbers of clones rather rapidly. But for this technique we require two different antibodies, which bind to two distinct domains of the desired protein, and this protein must not be produced by the nontransformed host cells. FACS. In case of animal cells, an automated system, called fluorescence activated cell sorter (FACS), can be used for very rapid (upto 1,000 cells/sec) sorting of transformed cells. This is applicable to all the genes whose products become arranged on the cell surface and are available for binding of specific antibodies. Therefore, these proteins must not be produced by the nontransformed host cells. The antibody molecules are attached to a fluorescent molecule and the transformed cells are treated with this antibody specific for the desired protein. The cells containing on their surface the protein in

question will interact with the fluorescent antibodies. Cells are then passed one by one in a stream between a laser and a fluorescence detector. The cells which fluoresce are deflected into a microculture tray, while the nonfluorescing cells are drawn away by an aspirator. This approach is also applicable to the genes encoding receptor proteins present on the cell surface; in such cases, fluorescent ligands (the concerned molecule to which the receptor binds) are used in the place of fluorescent antibodies.

Cell culture
growth factors which promotes cell proliferation, cell attachment and adhesion factors. Serum is obtained from human adult blood, placental, cord blood, horse blood, calf blood. The other forms of biological fluids used are coconut water, amniotic fluid, pleural fluid, insect haemolymph serum, culture filtrate, aqueous humour, from eyes etc.

iii) Tissue extracts for example Embryo extracts- Extracts from tissues such as embryo, liver, spleen, leukocytes, tumour, bone marrow etc are also used for culture of animal cells . Synthetic media Syntheic media are prepared artificially by adding several organic and inorganic nutrients, vitamins, salts, serum proteins, carbohydrates, cofactors etc. Different types of synthetic media can be prepared for a variety of cells and tissues to be cultured. Synthetic media are of two types- Serum containing media (media containing serum) and serum- free media (media with out serum). Examples of some media are: minimal essential medium (MEM), RPMI 1640 medium, CMRL 1066, F12 etc. Advantages of serum in culture medium are: i) serum binds and neutralizes toxins, (ii) serum contains a complete set of essential growth factors, hormones, attachment and spreading factors, binding and transport proteins, (iii) it contains the protease inhibitors, (iv) it increases the buffering capacity, (v) it provides trace elements. Disadvantages of serum in culture medium are: (i) it is not chemically defined and therefore its composition varies a lot, (ii) it is sometimes source of contamination by viruses, mycoplasma, prions etc, (iii) it increases the difficulties and cost of down stream processing, (iv) it is the most expensive component of the culture medium. 4) pH- Most media maintain the pH between 7 and 7.4. A pH below 6.8 inhibits cell growth. The optimum pH is essential to maintain the proper ion balance, optimal functioning of cellular enzymes and binding of hormones and growth factors to cell surface receptors in the cell cultures. The regulation of pH is done using a variety of buffering systems. Most media use a bicarbonate-CO2 system as its major component.

5) Osmolality- A change in osmolality can affect cell growth and function. Salt, Glucose and Amino acids in the growth media determine the osmolality of the medium. All commercial media are formulated in such a way that their final osmolality is around 300 mOsm. CELL BASED THERAPY The animal cell culture techniques are used in replacing the damaged and dead cells with normal and healthy cells using the stem cell technology. This therapy is called Cell-Based therapy which involves the use of stem cell technology involving the replacement of damaged and dead cells with normal and healthy cells. This is used to treat blood cancer, and other neuro-degenerative diseases etc. APPLICATIONS OF ANIMAL CELL CULTURE The animal cell cultures are used for a diverse range of research and development. These areas are: a) production of antiviral vaccines, which requires the standardization of cell lines for the multiplication and assay of viruses. b) Cancer research, which requires the study of uncontrolled cell division in cultures.

c) Cell fusion techniques.

d) Genetic manipulation, which is easy to carry out in cells or organ cultures.

e) Production of monoclonal antibodies requires cell lines in culture.

f) Production of pharmaceutical drugs using cell lines.

g) Chromosome analysis of cells derived from womb.

h) Study of the effects of toxins and pollutants using cell lines.

i) Use of artificial skin.

j) Study the function of the nerve cells. Somatic Cell Fusion One of the applications of animal cell culture is the production of hybrid cells by the fusion of different cell types. These hybrid cells are used for a the following purposes: (i) study of the control of gene expression and differentiation, (ii) study of the problem of malignancy, (iii) viral application,

(iv) gene mapping, (v) production of hybridomas for antibody production. In 1960s, in France for the first time, the hybrid cells were successfully produced from mixed cultures of two different cell lines of mouse. Cells growing in culture are induced by some of the viruses such as Sendai virus to fuse and form hybrids. This virus induces 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. Some other chemicals like polyethylene glycol also induce somatic cell fusion. Many commercial proteins have been produced by animal cell culture and there medical application is being evaluated. FIG SHOWING THE PRODUCTION OF T-PA Tissue Plasminogen activator (t-PA) was the first drug that was produced by the mammalian cell culture by using rDNA technology. The recombinant t-PA is safe and effective for dissolving blood clots in patients with heart diseases and thrombotic disorders.

When Monoclonal antibodies are used as enzymes using the technique of enzyme engineering, then they are calledabz y mes. Using animal cell cultures, it is also possible to produce Polyclonal Antibodies. Polyclonal antisera are derived from many cells therefore contains heterogeneous antibodies that are specific for several epitopes or an antigen. SCALE-UP OF ANIMAL CELL CULTURE

Modifying a laboratory procedure, so that it can be used on an industrial scale is called scaling up. Laboratory procedures are normally scaled up via intermediate models of increasing size. The larger the plant, the greater the running costs, as skilled people are required to monitor and maintain the machinery.The first prerequisite for any large scale cell culture system and its scaling up is the establishment of a cell bank. Master cell banks (MCB) are first established and they are used to develop Master Working Cell Banks (MWCB). The MWCB should be sufficient to feed the production system at a particular scale for the predicted life of the product. The cell stability is an important criteria so MWCB needs to be repeatedly subcultured and each generation should be checked for changes. A close attention should be paid to the volume of cultured cells as the volume should be large enough to produce a product in amounts which is economically viable. The volume is maintained by a) increasing the culture volume, (b) by increasing the concentration of cells in a reactor by continuous perfusion of fresh medium, so that the cells keep on increasing in number without the dilution of the medium. A fully automated bioreactor maintains the physicochemical and biological factors to optimum level and maintains the cells in suspension medium. The most suitable bioreactor used is a

compact-loop bioreactor consisting of marine impellers. The animal cells unlike bacterial cells, grow very slowly. The main carbon and energy sources are glucose and glutamine. Lactate and ammonia are their metabolic products that affect growth and productivity of cells. So, the on-line monitoring of glucose, glutamate, and ammonia is carried out by on line flow injection analysis (FIA) using gas chromatography (GC), high performance liquid chromatography (HPLC) etc. In batch cultures, mainly Roller Bottles with Micro Carrier Beads (for adherent cells) and spinner flasks (for suspension cultures) are used in Scale-up of animal cell culture process. Roller Bottles The Roller bottles provide total curved surface area of the micro carrier beads for growth. The continuous rotation of the bottles in the CO2 incubators helps to provide medium to the entire cell monolayer in culture.The roller bottles are well attached inside a specialized CO2 incubators. The attachments rotate the bottles along the long axis which helps to expose the entire cell monolayer to the medium during the one full rotation. This system has the advantage over the static monolayer culture: (a) it provides increase in the surface area, (b)

provides constant gentle agitation of the medium, (c) provides increased ratio of surface area of medium to its volume, which allows gas exchange at an increased rate through the thin film of the medium over the cells. Typically, a surface area of 750-1500 cm2 with 200500 ml medium will yield 1-2x108cells.

DIAGRAM SHOWING THE ROLLER BOTTLE CELL CULTURE

Micro Carrier Beads Micro carrier beads are small spherical particles with diameter 90-300 micrometers, made up of dextran or glass. Micro Carrier beads, increase the number of adherent cells per flask. These dextran or glass-based beads come in a range of densities and sizes. The cells grow at a very high density which rapidly exhausts the medium and therefore the medium has to be replaced for the optimum cell growth. At the recommended concentration when the microcarriers are suspended they provide 0.24 m2 area for every 100 ml of culture flask. Spinner cultures The spinner flask, was originally developed to provide the gentle stirring of microcarriers but are now used for scaling up the production of suspension cells. The flat surface glass flask is fitted with a Teflon paddle that continuously turns and agitates the medium. This stirring of the medium improves gas exchange in the cells in culture. The spinner flask used at commercial scale consists of one or more side arms for taking out samples and decantation as well. TYPES OF CELL CULTURES Primary cell culture

The maintenance of growth of cells dissociated from the parental tissue (such as kidney, liver) using the mechanical or enzymatic methods, in culture medium using suitable glass or plastic containers is called Primary Cell Culture. The primary cell culture could be of two types depending upon the kind of cells in culture. a) Anchorage Dependent /Adherent cells- Cells shown to require attachment for growth are set to be Anchorage Dependent cells. The Adherent cells are usually derived from tissues of organs such as kidney where they are immobile and embedded in connective tissue. They grow adhering to the cell culture. b) Suspension Culture/Anchorage Independent cells - Cells which do not require attachment for growth or do not attach to the surface of the culture vessels are anchorage independent cells/suspension cells. All suspension cultures are derived from cells of the blood system because these cells are also suspended in plasma in vitro e.g. lymphocytes. Secondary cell cultures When a primary culture is sub-cultured, it becomes known as secondary culture or cell line. Subculture (or passage) refers to the transfer of cells from one culture vessel to another culture vessel. Subculturing- Subculturing or splitting cells is required to periodically provide fresh nutrients and growing space for continuously growing cell lines. The process involves removing the

growth media, washing the plate, disassociating the adhered cells, usually enzymatically. Such cultures may be called secondary cultures. Cell Line A Cell Line or Cell Strain may be finite or continuous depending upon whether it has limited culture life span or it is immortal in culture. On the basis of the life span of culture, the cell lines are categorized into two types: a) Finite cell Lines - The cell lines which have a limited life span and go through a limited number of cell generations (usually 20-80 population doublings) are known as Finite cell lines.

CHARACHTERIZATION OF CELL LINES The cell lines are characterized by their a) growth rate and b) karyotyping. a) Growth Rate - A growth curve of a particular cell line is established taking into consideration the population doubling time, a lag time, and a saturation density of a particular cell line. A growth curve consist of: 1) Lag Phase: The time the cell population takes to recover from such sub culture, attach to the culture vessel and spread. 2) Log Phase: In this phase the cell number begins to increase exponentially. 3) Plateau Phase: During this phase, the growth rate slows or stops due to exhaustion of growth medium or confluency. b)Kar yo ty pi ng - Karyotyping is important as it determines the species of origin and determine the extent of gross chromosomal changes in the line. The cell lines with abnormal karyotype are also used if they continue to perform normal function. Karyotype is affected by the growth conditions used, the way in which the cells are subcultured and whether or not the cells are frozen. c) There are certain terms that are associated with the cell lines. These are as follows: (i) Split ratio- The divisor of the dilution ratio of a cell culture at subculture. (ii) Passage number- It is the number of times that the culture has been cultured.,

(iii) Generation number- It refers to the number of doublings that a cell population has undergone.
TABLE-SOME ANIMAL CELL LINES AND THE PRODUCTS OBTAINED FROM THEM

Cell line Product Human tumour Angiogenic factor Human leucocytes Interferon Mouse fibroblasts Interferon Human Kidney Urokinase Transformed human kidney cell line, TCL-598 Single chain urokinase-type plasminogen activator (scu-PA) Human kidney cell (293) Human protein (HPC) Dog kidney Canine distemper vaccine Cow kidney Foot and Mouth disease (FMD) vaccine Chick embryo fluid Vaccines for influenza, measles and mumps Duck embryo fluid Vaccines for rabies and rubella Chinese hamster ovary (CHO) cells 1. Tissue-type plasminogen activator (t-PA) 2. B-and gamma interferons

The culture animal material is washed in balanced salt solution to avoid contamination. The tissue to be cultured should be properly sterilized with 70% ethanol and removed surgically under aseptic conditions.

Disaggregation of tissue To obtain the cell suspension for primary cell culture, the tissue is disintegrated either mechanically or by using enzymes. (i) Physical or mechanical disaggregation- After removing the tissue under aseptic conditions, it is pressed through a sieve of 100 micrometer. It is then kept in a sterile Petri dish containing buffered medium with balanced salt solution. The cells are then alternately passed through the sieve of decreasing pore size (50 micrometer and 20 micrometer mesh). The debris which remains on the sieve is discarded and the medium containing cells is collected and cells are counted by using haemocytometer. This method is cheap and quick but it damages a lot of cells. (ii) Enzymatic disaggregation- In this method, enzymes are used for dislodging the cells of tissues. The two important enzymes used in tissue disaggregation are-collagenase and trypsin. -a) Collagenase- The intracellular matrix contains collagen therefore collagenase is used for disaggregation of embryonic, normal as well as malignant tissues. The tissues are kept in medium containing antibiotics and then dissected into pieces in basal salt solution. After washing the chopped tissue with distilled water, it is transferred to complete medium containing collagenase. After a few days (around 5 days), the mixture is pipetted so

that the medium gets dispersed. The whole treatment is left for sometimes during which the epithelial cells settle on bottom of test tubes. The enzyme collagenase is removed by centrifugation. Suspension consists of cells which are then plated out on the medium. (b) TrypsinUse of trypsin for disaggregation is called trypsinization. On the basis of role of temperature on trypsin, the activity of trypsin is of two types- Cold trypsinization and warm trypsinization. Cold trypsinization- The sample tissue to be disaggregated is chopped into 2-3 small pieces and kept in sterile glass vial. The tissues are subsequently washed with sterile water and dissected and then kept in BSS. The whole content is then placed on ice and soaked in cold trypsin for 4-6 hours to allow the penetration of enzymes in tissue. After this the trypsin is removed and the tissue is incubated at 36.50C for 20-30 minutes. About 10 ml of medium containing serum is added to the vials containing the cells and the cells are dispersed by repeated pipetting. The cells are counted by haemocytometer and are plated and incubated for 48-72 hours for cell growth.

Warm trypsinization- The initial steps are the same as in cold trypsinization however, in this case the tissue pieces are treated with warm trypsin (36.50C). The tissues are stirred for 4 hours and then pieces are allowed to settle down. The disassociated cells are collected at every 30 minutes. The process is repeated by adding fresh trypsin back to pieces and incubating the contents. The trypsin is removed by centrifugation after 3-4 hours during which the complete disaggregation of tissues takes place. The glass vials containing dispersed cells are then placed on ice. The cells are counted using haemocytometer and cell density is maintained at an appropriate number. The cells are then plated on medium and incubated for 48-72 hours for cell growth. (iii) Treatment with chelating agents- The tissues like epithelium (which needs Ca2+ and Mg2+ ions for its integrity are treated with chelating agents such as citrate and ethylene- diamine-tetra-acetic acid (EDTA). Chelating agents are mainly used for production of cell suspensions from established cultures of epithelial type. STEM CELL TECHNOLOGY Stem cells retain the capacity to self renew as well as to produce progeny with a restricted mitotic potential and restricted range of distinct types of differentiated cell they give rise to. The formation of blood cells also called haematopoiesis is the classical example of

concept of stem cells. Indirect assay methods were developed to identify the haematopoietic stem cells. The process ofhaematopoeis is occurs in the spleen and bone marrow in mouse. In human beings about 100,000 haematopoietic stem cells produce one billion RBC, one billion platelets, one million T-cells, one million B cells per kg body weight per day. Several methods have been developed to study haematopoiesis and stem cells: a) Repopulation assay- Edmens Snells group created mice which were genetically identical by mating of sibling mice after 21 generations. Two groups of mice were lethally X- irradiated to destroy their blood cell forming capacity. One of this group was injected with marrow cells from the femur bone of a normal and healthy albino mice. It was observed that this group survived whereas the mice in the other group died. The spleen of mice which survived had the colonies of the bone marrow cells just like bacterial colonies on a Petri plate. This came to be known as colony forming units of spleen (CFU-S) and the technique is known as repopulation assay. b) The in vitro clonal assay- In this assay, the stem cells proliferate to form colonies of

differentiated cells on semi-solid media. This assay helps in identifying growth factors required for the formation of blood cells from the primitive stem cells. One of the first commercialized

Genetic Engineering of animal cells and their applications The mammalian cells are genetically modified by introducing the genes needed for specific purposes such as production of specific proteins or to improve the characteristics of a cell line. The methods used to introduce the foreign genes/DNA into mammalian cells are: Electroporation, Lipofection, Microinjection and/or fusion of mammalian cells

with bacteria or viruses. After the integration of the foreign DNA into the mammalian cells, the transfected/transformed cells are selected by using suitable markers. Some of such markers in use are: Viral thymidine kinase, Bacterial dihydrofolate reductase, Bacterial neomycin phosphotransferase. It has been possible to overproduce several proteins in mammalian cells through genetic manipulations e.g. tissue plasminogen activator, erythropoietin, interleukin-2, interferon- beta, clotting factors VIII and IX, tumor necrosis factors. The recombinant mammalian cells are also conveniently used for the production of monoclonal antibodies. Manipulation of Gene Expression in Eukaryotes The eukaryotic organisms have the capability to bring about the post-translational modifications such as glycosylation, phosphorylation, proteolytic cleavage etc which ultimately helps in the production of stable and biologically active proteins. Due to these reasons the use of eukaryotic expression system is preferred however it is difficult to conduct experiments with eukaryotic cells. The introduction of a foreign DNA into animal cells is called transfection. The insert DNA in the eukaryotic cells may be associated with vector or integrated into the host

chromosomal DNA. Among the various hosts used for the expression of cloned genes, the common yeast Saccharomyces cerevisiae is the most extensively used. Besides this, the cultured insect cells are in use for expressing cloned DNAs. Baculoviruses exclusively infect insect cells. The DNA of these viruses encode for several products and their productivity in cells is very high to the extent of more than 10,000 times compared to mammalian cells. The baculoviruses not only carry a large number of foreign genes but can also express and process the products formed. By using baculovirus as an expression vector system, a good number of mammalian and viral proteins have been synthesized. The most commonly used baculovirus is Autographa californica multiple nuclear polyhedrosis virus (AcMNPV). It grows on the insect cell lines and produce high levels of polyhedrin or a recombinant protein. The mammalian cell expression vectors are used for the production of specific recombinant proteins and to study the function and regulation of mammalian genes. However, large-scale production of recombinant proteins with engineered mammalian cells is costly. The mammalian vector contains a eukaryotic origin of replication from an animal virus such as

Simian virus 40 (SV 40) and a prokaryotic origin of replication. It has a multiple cloning site and a selectable marker gene, both of which remain under the control of eukaryotic promoter and polyadenylation sequences. These sequences are obtained from either animal viruses (SV40, herpes simplex virus) or mammalian genes (growth hormone, metallothionein). The promoter sequences facilitate the transcription of cloned genes (at the multiple cloning site) and the selectable marker genes. On the other hand, the polyadenylation sequences terminate the transcription. Collection and purification process of Recombinant proteins As the recombinant proteins start accumulating in the host cells, it becomes important to collect and purify them. This is a tricky process since many times the recombinant protein is a foreign body for the host cells and the enzyme machinery of the host cell becomes activated to degrade the outside protein. One of the strategies adopted is the use of bacterial strains deficient in proteases or alternatively, the recombinant proteins are fused with the native host proteins. The fusion proteins are resistant to protease activity. Sometimes, the foreign proteins accumulate as aggregates in the host organism which minimizes the

protease degradation. The best way out is to quickly export and secrete out the recombinant proteins in to the surrounding medium. The recovery and the purification of foreign proteins is easier a) Gel and sponge technique- In this method, the gel (collagen) or sponges (gelatin) are used which provides the matrix for the morphogenesis and cell growth. The cells penetrate these gels and sponges while growing. b) Hollow fibers technique- In this method, hollow fibers are used which helps in more efficient nutrient and gas exchange. In recent years, perfusion chambers with a bed of plastic capillary fibers have been developed to be used for histotypic type of cultures. The cells get attached to capillary fibers and increase in cell density to form tissue like structures. c) Spheroids The re-association of dissociated cultured cells leads to the formation of cluster of cells called spheroids. It is similar to the reassembling of embryonic cells into specialized structures. The principle followed in spheroid cultures is that the cells in heterotypic or homotypic aggregates have the ability to sort themselves out and form groups which form tissue like architecture. However, there is a limitation of diffusion of nutrients and gases in these cultures.

d) Multicellular tumour spheroids- These are used as an in vitro proliferating models for studies on tumour cells. The multicellular tumour spheroids have a three dimensional structure which helps in performing experimental studies related to drug therapy, penetration of drugs besides using them for studying regulation of cell proliferation, immune response, cell death, and invasion and gene therapy. A size bigger than 500 mm leads to the development of necrosis at the centre of the MCTS. The monolayer of cells or aggregated tumour is treated with trypsin to obtain a single cell suspension. The cell suspension is inoculated into the medium in magnetic stirrer flasks or roller tubes. After 3-5 days, aggregates of cells representing spheroids are formed. Spheroid growth is quantified by measuring their diameters regularly. The spheroids are used for many purposes. They are used as models for a vascular tumour growth. They are used to study gene expression in a threedimensional configuration of cells. They are also used to study the effect of cytotoxic drugs, antibodies, radionucleotides, and the spread of certain diseases like rheumatoid arthritis. Organotypic cultures

These cultures are used to develop certain tissues or tissue models for example skin equivalents have been created by culturing dermis, epidermis and intervening layer of collagen simultaneously. Similarly models have been developed for prostrate, breast etc. Organotypic culture involves the combination of cells in a specific ratio to create a component of an organ. BIOETHICS IN ANIMAL GENETIC ENGINEERING There are some serious issues related to genetic modification of animals using animal genetic engineering techniques. One is not sure of the consequences of these genetic modifications and the further interaction with the environment. Proper clinical trials are also necessary before one can use it for commercial purposes. In the recent past people have raised objections on some of the methods used e.g. the transfer of a human genes into food animals, use of organisms containing human genes as animal feed. Some religious groups have expressed their concern about the transfer of genes from animals whose flesh is forbidden for use as food into the animals that they normally eat. Transfer of animal genes into food plants that may be objectionable to the vegetarians. Besides this, there are several other aspects of this issue have to be sorted out.

a) What will be the consequences, if a modified animal will breed with other domestic or wild animals thereby transferring the introduced genes to these populations? b) What are the health risks to human on consumption of genetically modified animals and

their products? c) With the production of disease resistant animals, what will be the effect on ecology? d) There is also wide spread concern about the risks of human recipients getting infected with animal viral diseases after a xenotransplantation., which might infect the population at large. e) There are also concerns about the risk that drug resistance gene markers used in genetic engineering procedures might inadvertently be transferred and expressed. The need of the hour is to formulate clear guidelines which should be followed while using genetic engineering techniques in bio-medical research. e.g. products from transgenic organisms should be clearly marked to give choice to people who follow dietary restrictions due to religious beliefs. In fact all the ethical and moral issues raised by some aspects of biotechnology should be addressed by open discussion and dialogue.

CELL AND TISSUE ENGINEERING Tissue engineering refers to the application of the principles of engineering to cell culture for the construction of functional anatomical units- tissues/organs. The aim of tissue engineering is nothing but to supply the various body parts for the repair or replacement of damaged tissues or organs. It is now possible to grow skin cells, blood cells cardiac cells etc. by using the ability of stem cells to proliferate and differentiate. During the last decade, the tissue culture work in animals demonstrated that virtually any human tissue or organ can be grown in culture. This became possible only after it became known that the ability of cultured cells to undergo differentiation can be restored. Skin was the first organ to be cultured in artificial media and could be successfully used for transplantation following serious skin burns. For past few years some of the biotech companies like ATS (Advanced Tissue Science, USA), Biosurface Technology (BTI, Cambridge) and Organogenesis, are developing artificial skins to the stage of clinical trials. In the field of tissue replacement, focus of attention is the Artificial cartilage. As it is not vascularized, it is not rejected due to immunogenic response. This will have lots of

implications in the treatment of sport related injuries and diseases like arthritis. Design and engineering of tissues The design and tissue engineering should essentially cause minimal discomfort to the patient. The damaged tissues should be easily fixed with the desired functions quickly restored. Another important factor controlling the designing of tissue culture is the source of donor cells. The cells from the patient himself, is always preferred as it considerably reduces the immunological complications. However under certain situations allogeneic cells (cells taken from a person other than the patient) are also used. The other important factors are the support material, its degradation products, cell adhesion characteristics etc. It was demonstrated in 1975 that human keratinocytes could be grown in the laboratory in a form suitable for grafting. A continuous sheet of epithelial cells can be grown now however there is still difficult to grow TE skin with the dermal layer with all the blood capillaries, nerves, sweat glands, and other accessory organs. Some of the implantable skin substitutes which are tissue engineering skin constructs with a limited shelf life of about 5 days are:

a) Integra TM A bioartificial material composed of collagen-glycosaminoglycan and is mainly used to carry the seeded cells. b) DermagraftTM- This is composed of poly glycolic acid polymer mesh seeded with human

dermal fibroblasts from neonatal foreskins. c) ApligrafTM- It is constructed by seeding human dermal fibroblasts into collagen gel with the placement of a layer of human keratinocytes on the upper surface. These tissue constructs integrate into the surrounding normal tissue and form a good skin cover with minimum immunological complications. The urothelial cells and smooth muscle cells from bladder are now being cultured and attempts are on to construct TE urothelium. Some progress has also been made in the repair of injured peripheral nerves using tissue engineered peripheral nerve implants. The regeneration of the injured nerve occurs from the proximal stump to rejoin at distal stump. The regeneration process requires substances like(a) Conduct material- The conduct material is composed of collagenglycosaminoglycans, PLGA (poly lactic- co- glycolicacid), hyaluronan and fibronectin and forms the outer layer. (b) Filling material- The filling material contains collagen, fibrin, fibronectin and agarose. This supports the neural cells for regeneration. and

(c) Additives- A large number of other factors are also added e.g. growth factors, neurotrophic factors such as fibroblast growth factor (FGF), nerve growth factor (NGF). The other important applications of tissue engineering are in gene therapy, pseudo-organs and as model cell systems for developing new therapeutic approaches to human diseases.The attempts are on to create tissue models in the form of artificial organs using tissue engineering. The artificial liver is being created using hepatocytes cultured as spheroids and held suspended in artificial support system such as porous gelatin sponges, agarose or collagen. Some progress has been made in the area of creating the artificial pancreas using spheroids of insulin secreting cells which have been developed from mouse insulinoma beta cells. Three dimensional brain cell cultures have been used for the study of neural myelination, neuronal regeneration, and neurotoxicity of lead. The aggregated brain cells are also being used to study Alzheimers disease and Parkinsons disease. Thyroid cell spheroids are being used to study cell adhesion, motility, and thyroid follicle biogenesis. (Table 8.2 page 155, gupta)
TABLE DEPICTING THE TECHNOLOGICAL GOALS AND AREAS OF RESEARCH IN TISSUE ENGINEERING

Growth of cells in three- dimensional systems

Delivery systems for protein therapeutics Cell cultivation methods for culturing recalcitrant cells Expression of transgenic proteins in transplantable cells To develop vehicles for delivering transplantable cells Development of markers for tracking transplanted cells Avoiding immunogenicity in transplantable cells Development of in vivo and ex vivo biosensors for

monitoring cell behaviour during tissue production DOWNSTREAM PROCESSING Downstream processing or downstreaming is the extraction and purification of the desired end products of fermentation processes. Such products might include cells, solvents or solutes. Various processes are available for the separation of cells from the fermentation broth in which they are grown, including flocculation, filtration, centrifugation, sedimentation or flotation. The procedure adopted depends on whether it is the cells, or the solution surrounding them, that contains the desired end products.