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Animal Biotechnology Cheat Sheet

Over 1000 words strictly aligned with the DBT BET syllabus. Master Hybridoma Technology, Cell Culture Media, Stem Cells, and Transgenics to maximize your Section B scores.

1. Animal Cell Culture: The Foundation

Culturing animal cells is vastly different and more complex than plant tissue culture. Animal cells lack cell walls, are highly prone to mechanical shearing, and require complex growth factors.

Media and Serum Constituents

The basal medium (like DMEM or RPMI) contains glucose, amino acids, and vitamins. However, the most crucial additive is Fetal Bovine Serum (FBS). FBS provides:

• Growth Factors: PDGF, EGF which stimulate cell division.
• Adhesion Factors: Fibronectin and Vitronectin, essential for Anchorage-dependent cells to stick to the plastic flask.
• Protease Inhibitors: Like α-1 antitrypsin, which stops trypsin from over-digesting cells during subculturing.

Phenol Red Indicator: Used in culture media to monitor pH.
• Normal pH (7.4): Red/Pink.
• Acidic (low pH): Yellow (Indicates bacterial contamination or metabolic exhaustion).
• Basic (high pH): Purple (Indicates fungal contamination or lack of CO2).

Cell Line Characteristic	Primary Cell Culture	Continuous (Immortalized) Cell Line
Lifespan	Finite (undergoes senescence after ~50 divisions, Hayflick limit).	Infinite (Evades apoptosis, usually has active Telomerase).
Contact Inhibition	Yes. They stop dividing when they touch each other (form a monolayer).	No. They pile up on top of each other.
Anchorage Dependence	Yes. Must attach to a solid surface to survive.	No. Can often be grown in suspension.
Examples	Fibroblasts, Primary Hepatocytes, Epithelial cells.	HeLa (Cervical cancer), CHO (Chinese Hamster Ovary).

2. Hybridoma Technology (Monoclonal Antibodies)

Developed by Kohler and Milstein, this is arguably the most repeatedly asked topic in the DBT BET exam. The goal is to produce large quantities of highly specific Monoclonal Antibodies (mAbs) against a single epitope.

The Two Nucleotide Synthesis Pathways

To understand selection, you must know how cells make nucleotides:

• De Novo Pathway: Builds nucleotides from scratch using sugars and amino acids. It is completely blocked by the drug Aminopterin.
• Salvage Pathway: Recycles free bases (Hypoxanthine and Thymidine) to make nucleotides. This requires the enzymes HGPRT (for purines) and TK (for pyrimidines).

The Selection Mechanism in HAT Medium

We fuse a mortal B-cell (which produces antibodies) with an immortal Myeloma cancer cell (which divides endlessly). The myeloma cell line used is strictly genetically engineered to be HGPRT negative (-).

After fusion, the cells are grown in HAT Medium (Hypoxanthine, Aminopterin, Thymidine):

• Unfused Myeloma cells die: Aminopterin blocks their De novo pathway. Since they lack HGPRT, they cannot use the Salvage pathway either. They die of nucleotide starvation.
• Unfused B-cells die: They have HGPRT, but they are mortal and die naturally in culture after a few days.
• Hybridoma Cells SURVIVE: They get immortality from the Myeloma parent and the HGPRT gene from the B-cell parent, allowing them to use the Salvage pathway and thrive in HAT medium.

3. Transgenics & Cloning (SCNT)

Animal cloning broke scientific barriers with the creation of Dolly the Sheep in 1996 using Somatic Cell Nuclear Transfer (SCNT).

Fig: Somatic Cell Nuclear Transfer (SCNT) Mechanism

How SCNT Works:

1. Extract an unfertilized egg (oocyte) and remove its haploid nucleus (Enucleation).
2. Take a somatic cell (e.g., skin or mammary gland cell) from the animal to be cloned.
3. Fuse the diploid somatic nucleus with the enucleated egg cell using a mild electrical pulse.
4. The egg cytoplasm "reprograms" the somatic nucleus back to a totipotent state.
5. The developing embryo is implanted into a surrogate mother.

4. Stem Cells & Advanced Genetic Engineering

Stem cell biology and gene editing tools like Cre-LoxP are heavily featured in the applied sections of the paper.

Stem Cell Potency

• Totipotent: Can form every cell in the body PLUS the extraembryonic tissues (placenta). Example: The Zygote (up to the 8-cell stage).
• Pluripotent: Can form all three germ layers (ecto, meso, endoderm), but NOT the placenta. Example: Embryonic Stem Cells (ESCs) derived from the Inner Cell Mass of the blastocyst.
• Multipotent: Restricted to a specific lineage. Example: Hematopoietic stem cells (can make all blood cells, but not brain cells).

Induced Pluripotent Stem Cells (iPSCs): Shinya Yamanaka won the Nobel Prize for discovering that adult somatic cells (like fibroblasts) can be reprogrammed back to a Pluripotent Embryonic state by introducing 4 specific transcription factors: Oct4, Sox2, c-Myc, and Klf4 (The Yamanaka Factors).

Cre-LoxP Recombination (Conditional Knockout)

Standard gene knockouts can be lethal to the embryo. The Cre-LoxP system allows researchers to knock out a gene only in a specific tissue or at a specific time. Cre is a recombinase enzyme derived from a bacteriophage. LoxP sites are short DNA sequences. When Cre is expressed, it cuts and recombines the DNA located exactly between two LoxP sites, deleting the gene.

Guaranteed Exam Hits

PYQ Direct Statements (Ye questions aayenge hi aayenge!)

• Gene Pharming (Tracy the Sheep): Transgenic animals are used as bioreactors. Tracy the sheep was genetically engineered to produce human α-1-antitrypsin in her milk, a protein used to treat Emphysema (lung disease) and Cystic Fibrosis.
• OncoMouse (Harvard Mouse): The first patented transgenic animal. It carries an activated mutant oncogene (v-Ha-ras) which makes it highly susceptible to cancer, making it an invaluable model for oncology research.
• SCID Gene Therapy: The first successful human gene therapy was performed on a 4-year-old girl with Severe Combined Immunodeficiency (SCID). She was given the functional gene for the enzyme Adenosine Deaminase (ADA) using a retroviral vector.
• Trypsinization in Culture: Trypsin is a proteolytic enzyme used to detach adherent cells from the flask during subculturing. It requires Calcium to be removed, which is why we wash the cells with EDTA (a calcium chelator) before adding trypsin.
• Microinjection Specificity: When generating transgenic mice via direct DNA microinjection, the recombinant DNA must be injected specifically into the Male Pronucleus of the fertilized egg, because it is physically larger and closer to the surface than the female pronucleus.
• Polyclonal vs Monoclonal: Polyclonal antibodies recognize multiple different epitopes on a single antigen (made by different B-cell clones). Monoclonal antibodies recognize only ONE specific epitope on an antigen (made by a single cloned B-cell line).

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