Cell Signalling 3 Flashcards
What is transient transfection ?
Definition: Introduction of foreign DNA (or RNA) into a cell where the genetic material does not integrate into the host genome.
Duration: Short-term expression, usually lasting 24–96 hours.
Mechanism: The introduced genetic material remains in the cytoplasm or nucleus but is not integrated into the genome.
Stability: Temporary and degrades over time as the plasmid DNA is diluted during cell division or destroyed.
Uses: Quick experiments like protein expression, reporter gene assays, or studying short-term effects of a gene.
Advantages: Fast, straightforward, and does not require selection processes.
Disadvantages: Expression is short-lived and may vary between cells
Outline a routine method for the insertion of cDNA into a plasmid vector (part 1)
cDNA Preparation:
Synthesize cDNA from mRNA using reverse transcriptase.
Restriction Enzyme Digestion:
Cut both cDNA and plasmid vector with compatible restriction enzymes to create sticky or blunt ends.
Ligation:
Mix digested cDNA and plasmid with DNA ligase to covalently join the DNA fragments.
Transformation:
Introduce recombinant plasmid into competent bacterial cells (e.g., via heat shock or electroporation).
Selection:
Plate transformed bacteria on selective media (e.g., containing antibiotics) to isolate colonies with the plasmid.
Screening:
Confirm insertion via colony PCR, restriction digestion, or sequencing.
Explain the value of tagging recombinant proteins
Simplifies protein purification
Aids protein identification
Improves protein solubility
Facilitates localisation studies
Assists in Protein-Protein Interaction Studies
Design approaches to discover the function of signalling proteins
Gene Knockout/Knockdown:
Remove or reduce protein expression (CRISPR, siRNA) to study functional loss.
Overexpression Studies:
Overexpress protein in cells to assess its role in signaling pathways.
Mutagenesis:
Introduce mutations to identify critical functional domains (e.g., phosphorylation sites).
Protein Interaction Studies:
Identify binding partners (e.g., co-immunoprecipitation, yeast two-hybrid).
Inhibitors and Activators:
Use small molecules or peptides to block/activate protein and assess pathway effects.
Reporter Assays:
Monitor downstream signaling using luciferase or fluorescent reporters.
Live Imaging:
Visualize protein localization and dynamics (e.g., GFP-tagged proteins).
What is stable transfection ?
Definition: Introduction of foreign DNA into a cell, followed by its integration into the host genome.
Duration: Long-term, as the inserted genetic material is replicated and passed to daughter cells during cell division.
Mechanism: The genetic material integrates into the host genome via recombination or other mechanisms.
Stability: Permanent, allowing consistent and reproducible expression of the gene of interest.
Uses: Long-term studies, including drug testing, protein production, and functional studies requiring stable gene expression.
Advantages: Reliable and consistent expression over many generations of cells.
Disadvantages: Time consuming due to the need for selection processes (e.g., antibiotic selection) to isolate stably transfected cells.
