Honours / Discipline Specific Elective (DSE)
ANIMAL CELL BIOTECHNOLOGY
►Unit 1: Introduction
Concept and Scope of Biotechnology
Q. What is the objective, vision, and mission of TDB?
A Technology Development Board (TDB) has been set up by the Government for the promotion of product development. The TDB works with universities, industries and the National institutes. The Technology information, Forecasting and Assessment Council (TIFAC) has prepared a ‘Vision 2020’ document which consists of biotechnology also. The objectives of TDB are— the objectives are to:
promote new ideas from small enterprises even at the risk of failures, encourage production of competitive consumer products, motivate industries and R&D institutions for product innovation, develop socially relevant and profitable technologies, identify and act in areas requiring strategic interventions, Invest in core Technological Strengths to Enable India Industry to Stand-up to the competitive Pressure and Become a global Player.
TDB’s goal is to make the weak zone of technology development and commercialization strong in selected sector by supporting technology development in the industry with short, medium and long term risk horizon. Technology priorities for India are:
(i)Raising capital productivity by modernising and reducing costs by constant technological inputs to the large base of basic infrastructure like energy, transport, housing, communication etc. though in the short term, some may have to resort to large scale imports of capital goods and know-how.
(ii)Technology support in areas which are currently driving export to enhance value-addition and to improve the quality, quantity and value of exports.
(iii)Technology support in selected small scale sector whose future in many ways depends on high technology development; and
(iv)Work on selected area of frontier market-oriented technologies at international scale and in which India can make an impact and attain a globally competitive edge.
►Unit 2: Techniques in Gene manipulation
Recombinant DNA technology, Restriction endonucleases. Cloning Vectors & their features: Plasmids, Phage vectors, Cosmids, Phagemids, BAC, YAC, and HAC. Shuttle and Expression Vectors. Construction of Genomic libraries and cDNA libraries Transformation techniques: Cloning in bacteria and detection technique of clone
Q. Define Neo-schizomer and Isoschizomer with examples.
Restriction enzymes with the same sequence specificity and cut site are known as isoschizomers. SphI (CGTAC/G) and BbuI (CGTAC/G) are isoschizomers of each other. The first enzyme discovered which recognizes a given sequence is known as the prototype; all subsequently identified enzymes that recognize that sequence are isoschizomers. Isoschizomers are isolated from different strains of bacteria and therefore may require different reaction conditions.
Enzymes that recognize the same sequence but cleave at different points, for example SmaI (CCC/GGG) and XmaI C/CCGGG), are sometimes known as neoschizomers.
Q. What is HAC?
Human artificial chromosomes (HACs) represent extrachromosomal gene delivery and gene expression vector system. Although this technology is less advanced than virus derived vectors, HACs have several potential advantages over currently used episomal viral vectors for gene therapy applications. First, the presence of a functional centromere provides long-term stable maintenance of HACs as single-copy episomes without integration to the host chromosomes. Second, there is no upper size limit to DNA cloned in a HAC: entire genomic loci with all regulatory elements can be used. Finally, HAC vectors minimize adverse host immunogenic responses and the risk of cellular transformation.
A human artificial chromosome (HAC) is a microchromosome that can act as a new chromosome in a population of human cells. That is, instead of 46 chromosomes, the cell could have 47 with the 47th being very small, roughly 6–10 megabases (Mb) in size instead of 50–250 Mb for natural chromosomes, and able to carry new genes introduced by human researchers. Ideally, researchers could integrate different genes that perform a variety of functions, including disease defence.
►Unit 3: Animal cell Culture
Basic techniques in animal cell culture and organ culture, Primary Culture and Cell lines, Culture media – Natural and Synthetic, Stem cells, Cryopreservation of cultures. Agarose and Polyacrylamide Gel Electrophoresis, Southern, Northern and Western blotting, Polymerase chain reaction: Allele specific, RAPD & RT PCR.
Q. What is ARMS-PCR? State its limitations
The allele-specific PCR is also called as the (amplification refractory mutation system) ARMS-PCR because of the use of two different primers for two different alleles. Here the word “refractory” is very important (Refractory= resistant to something). Two sets of primers are designed, the mutant set of the primer is refractory (resistant) to the normal PCR and the normal set of the primers are refractory to the mutant PCR reaction. That is why it is called an amplification refractory mutation system. The name ARMS-PCR is given by its actual developer C. R. Newton.
Limitations:
Deletion/other major duplication and chromosomal abnormalities cannot be detected, Only known SNPs are detected by ARMS-PCR, Internal control is required because of the chance of the false-negative results, It is temperature-sensitive. A minute fluctuation in the temperature leads to false-positive results, Thousands of SNPs cannot be detected in a single assay.
►Unit 4: Fermentation
Different types of Fermentation: Submerged & Solid state; batch, Fed batch & Continuous; Stirred tank, Air Lift, Fixed Bed and Fluidized. Downstream Processing: Filtration, centrifugation, extraction, chromatography, spray drying and lyophilization.
Q. What is the basic principle of fermentation?
In the absence of aerobic or anaerobic respiration, NADH is not oxidized by the electron transport chain because no external electron acceptor is available. Yet NADH produced in the glycolytic pathway during the oxidation of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate must still be oxidized back to NAD. If NADis not regenerated, the oxidation of glyceraldehyde 3-phosphate will cease and glycolysis will stop. Many microorganisms solve this problem by slowing or stopping pyruvate dehydrogenase activity and using pyruvate or one of its derivatives as an electron and hydrogen acceptor for the reoxidation of NADH in a fermentation process. This may lead to the production of more ATP. The process is so effective that some chemoorganoheterotrophs do not carry out respiration even when oxygen or another exogenous acceptor is available.
Reoxidation of NADH During Fermentation. NADH from glycolysis is reoxidized by being used to reduce pyruvate or a pyruvate derivative (X). Either lactate or reduced product Y result.
►Unit 5: Application in Health
Hybridoma technology, Production of recombinant Proteins: Insulin and growth hormones.
Q. What do you mean by HAT selection?
i)Fused cells are grown in HAT medium (named for its 3 components-hypoxanthine, aminopterin, and thymidine) in which neither of the parent cells can survive, but the A-B fusion cells can.
ii)HAT selection depends on the fact that mammalian cells can synthesize nucleotides by two different pathways-the de novo and the salvage pathways.
The de novo pathways, in which a methyl or formyl group is transferred from an activated form of tetrahydrofolate, is blocked by amniopterin, a folic acid analog. When the de novo pathway is blocked, cells utilize the salvage pathway, which bypasses the aminopterin block by converting purines and pyrimidines directly into nucleic acid.
iii)The enzymes catalysing the salvage pathway include hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and thymidine kinase (TK).
A mutation in either of these two enzymes blocks the salvage pathway. HAT medium contains aminopterin to block the de novo pathway and hypoxanthine and thymidine to allow growth via the salvage pathway. When two types of cells, one with a mutation in TK and the other with a mutation in HGPRT are fused only the hybrid cells will contain the full component of necessary enzymes for growth on HAT medium via the salvage pathway. Thus only hybrid cells will grow in HAT medium, unfused cells and homogeneous heterokaryons will not survive.