Techniques

Question 1

Which approaches are there when investigating ion channels?

Answer 1

1. Electrophysiology (TEVC, Patch clamping)
2. Biochemistry (Enzyme Assays, PPI/ Immunoprecipitation/Antibody assays e.g. western blotting and pull downs)
3. Imaging (Fluorescence Microscopy FRAP and FRET)
4. Structural Approaches (X-ray Crystallography, Cryo-Electron Microscopy/Cryo-EM)
5. Computational Approaches ( Molecular Dynamics (MD) Simulations and Bioinformatics
6. In vivo animal studies (Knock-out/-down)
7. In vitro cell studies (Cell Lines and Native cells)

Question 1

Explain knock-out

Answer 1

Knock-out (KO):
Involves completely eliminating the function of a target gene by disrupting its sequence. This is often achieved through homologous recombination or CRISPR-Cas9.

• Homologous Recombination: Involves replacing a target gene with a modified or non-functional copy.
• CRISPR-Cas9 KO: Utilizes the CRISPR system to introduce double-strand breaks in the gene, leading to its disruption during repair.

Question 2

Explain knock-down

Answer 2

Knock-down (KD):
Reduces the expression of a gene, typically through RNA interference (RNAi) or short hairpin RNA (shRNA) technologies.

• RNA Interference (RNAi): Introduction of small interfering RNA (siRNA) to degrade mRNA and reduce gene expression.
• Short Hairpin RNA (shRNA): Induces gene silencing through the expression of short RNA sequences.

Question 3

How does CRISPR work?

Answer 3

Components:
* CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats): DNA sequences derived from bacteria and archaea that store information about viruses.
* Cas9 (CRISPR-associated protein 9): An enzyme that acts as molecular scissors, cutting DNA at specific locations. Cas9 is responsible for cutting the DNA at the precise location specified by the gRNA.
* Guide RNA (gRNA): A synthetic RNA molecule designed to match a specific DNA sequence in the target organism. The gRNA acts as a guide to locate the target DNA sequence.

Mechanism:
* Recognition of Target DNA:
The gRNA is designed to complement a specific sequence of the target DNA. When the gRNA locates this sequence, it binds to the DNA.
* Formation of the gRNA-Cas9 Complex:
The gRNA guides the Cas9 protein to the exact location on the DNA where the target sequence is present. The Cas9 protein undergoes a conformational change, creating an active complex.
* DNA Cleavage:
Cas9 acts as molecular scissors and induces a double-strand break (DSB) at the targeted location on the DNA. The cell’s natural repair mechanisms, non-homologous end joining (NHEJ) or homology-directed repair (HDR), then kick in to fix the break.
* Repair Mechanisms:
Non-homologous End Joining (NHEJ): This is an error-prone repair process that may introduce insertions or deletions (indels) at the site of the break. This can result in gene disruption or knockout.
Homology-Directed Repair (HDR): If a template DNA with the desired sequence is provided along with the CRISPR components, the cell can use this template to repair the break, allowing precise editing of the targeted gene.

Applications:
* Gene Editing: Introducing precise changes in the genome.
* Gene Regulation: Modulating gene expression without altering the DNA sequence.

Question 4

What does Heterologous Expression mean?

Answer 4

Purpose: Expressing genes from one organism (source) in a different organism (host).

Applications:
- Protein Production: Producing proteins for research, therapeutic, or industrial purposes.
- Functional Studies: Understanding the role of specific genes in a different biological context.

Expression Systems:
- Bacterial Systems (e.g., E. coli): Suitable for simple proteins but may lack post-translational modifications.
- Yeast (e.g., Saccharomyces cerevisiae): Useful for eukaryotic protein expression with some post-translational modifications.
- Mammalian Cells (e.g., CHO cells): Ideal for expressing complex proteins with proper folding and post-translational modifications.

Challenges:
- Codon Usage: Differences in codon preferences between species may affect protein expression.
- Protein Toxicity: Overexpression of certain proteins can be harmful to host cells.

Question 5

Explain cell lines

Answer 5

Advantages:
- Homogeneity: Consistent genetic and phenotypic characteristics.
- Convenience: Ease of maintenance and scalability.

Examples:
- HeLa cells: Human cervical cancer cells widely used in research.
- HEK293 cells: Human embryonic kidney cells commonly used for transfection and protein expression.

Immortalization Techniques: Introduction of genes (e.g., telomerase) to extend the replicative lifespan of cells.

Native Cells:
- Primary Cells: Isolated directly from tissues and retain more in vivo characteristics.
- Organoids: Three-dimensional cell cultures that mimic organ structures and functions.
- Microfluidic Systems: Devices that mimic physiological conditions for studying cell behavior.