(a) What is transgenesis? Describe the methods and applications of transgenesis in animals. (20 marks) (b) Describe the principle…

(a) Transgenesis: methods and applications

Transgenesis is the stable introduction of foreign DNA (a transgene) into the genome of an animal, including its germline, so that it is expressed and inherited by the offspring.

Methods:

• Pronuclear microinjection: linear DNA is injected into the male pronucleus of a fertilised egg, and the egg is transferred to a foster mother. It is simple and widely used in mice, but integration is random and efficiency is low.
• Retroviral or lentiviral vectors: the transgene is carried into early embryos or cells; delivery is efficient but the insert size is limited and insertion can be harmful.
• Embryonic stem cell method: the construct is put into ES cells, the cells carrying it are selected (including gene targeting by homologous recombination), and injected into blastocysts to give chimeras; this allows precise changes.
• Nuclear transfer: nuclei from transgenic cultured cells are transferred into enucleated eggs, as in cloning of livestock.
• Genome editing (CRISPR–Cas9): targeted insertion, knock-out or knock-in at a chosen site with fewer random insertions, though off-target effects must be checked.
• Sperm-mediated gene transfer is another method.

Applications:

• Biopharming: human proteins in milk of transgenic animals, such as antithrombin from goats.
• Disease models: for example mice carrying human genes for cancer or Alzheimer's disease.
• Livestock improvement, such as faster-growing fish (AquAdvantage salmon).
• Research on gene function and toxicity testing; pigs modified as organ donors for xenotransplantation.
• Ornamental animals such as GloFish.

Ethical and biosafety issues need regulation.

(b) Polymerase chain reaction (PCR)

PCR is an in vitro method of making millions of copies of a chosen DNA segment from a very small amount of template. Principle: a thermostable DNA polymerase (Taq polymerase, from Thermus aquaticus) extends two synthetic primers that flank the target, in a buffer with Mg²⁺ and dNTPs. Each cycle has three steps in a thermocycler:

• denaturation at about 94–95 °C, separating the strands;
• annealing at about 50–65 °C, in which the primers bind to the complementary ends;
• extension at about 72 °C, in which the polymerase makes new strands.

The amount of the target doubles in each cycle, so n cycles give about 2ⁿ copies, and 25–35 cycles give a billion-fold amplification. Variants include reverse-transcription PCR (RNA to cDNA first) and real-time (quantitative) PCR.

Applications: diagnosis of infections (such as tuberculosis, HIV and COVID-19) and genetic diseases; DNA fingerprinting in forensics and paternity tests; gene cloning and sequencing; gene-expression studies; detection of mutations in cancer; and ancient DNA and wildlife identification.

(c) Learning and memory in animals

Learning is a relatively permanent change in behaviour due to experience, and memory is the storage and retrieval of what is learnt. Forms of learning, with examples:

• Habituation: a decreasing response to a harmless repeated stimulus, as in Aplysia gill withdrawal or a bird ignoring a scarecrow.
• Sensitisation: a stronger response after a strong stimulus.
• Imprinting: Lorenz's goslings following the first moving object seen after hatching.
• Classical conditioning: Pavlov's dogs salivating to a bell associated with food.
• Operant conditioning: Skinner's rat pressing a lever for food reward.
• Insight and latent learning, as in chimpanzees stacking boxes, or rats in a maze.

The neural basis is synaptic plasticity. Short-term memory involves changes in existing synapses; long-term memory needs gene expression (the transcription factor CREB) and new synapses, as shown by Kandel's work on Aplysia. In mammals, long-term potentiation in the hippocampus is important for spatial and declarative memory and the amygdala for emotional memory; damage to the hippocampus impairs the rat's learning of the platform in the Morris water maze.