Studying Cells Flashcards
What are some problems with primary cells?
Can be difficult to isolate, ethical issues, often difficult to genetically manipulate, will only grow for a limited number of generation in vitro.
What are cell lines usually derived from?
Tumours
What are the benefits of cell lines?
Can grow in the lab for many generations if not indefinitely, easily cultured and available, often more easily transfected.
Give an example of a cell line?
HeLa cells: a cervical carcinoma cell line derived from Henrietta Lacks tumour. Immortalised and used throughout the world as a model human cell line.
How are cells grown in culture?
In growth medium supplemented with serum to provide the required growth factors. Additional factors: cytokines, L-Glutamine, antibiotics, non-essential amino avoids can be added. Either I’m suspension or adhered to a plastic surface. All performed under a laminar flow hood.
Define transfection?
The process if introducing Nucleic avid into cells.
When is transfection required?
Protein of interest is not expressed, there’s is not an antibody to detect the protein so a tagged form of the protein is required to be expressed, you may wish to study a mutant form of a protein, you may want to analyse mutants in order to define sequences of the protein which are important for its function.
What are typical mammalian cell DNA expression constructs?
Typically DNA plasmids that can be propagated in E.coli and have selection markers.
How is the gene of interest inserted into the construct?
Electroporation to increase the membranes permeability, lipid-bases reagents encapsulate and fuse with the membrane or calcium phosphate to form a precipitate. All inserted downstream of promoters and tags are often added in order to detect expression.
What is RNA interference?
The process of knocking down gene expression using small interfering RNAs in a sequnce specific fashion.
How is RNA interference used to knock down gene expression?
Small double stranded interfering RNAs specific for the target gene are transfected into the cells. The anti-sense strand is bound by the RISC complex which cleaves mRNAs with a complementary sequence preventing translation.
What is immunoblotting?
Use of antibodies to detect the expression of proteins.
How does immunoblotting work?
Antibodies are either specific to the protein or it’s tag, proteins are separated by SDS-PAGE and a western blot is performed. The blot is then probed with the antibodies and detected by binding of a second antibody who’s tag can be visualised.
What is fluorescence microscopy used for?
To detect proteins tagged with fluorescent proteins.
What is the most commonly used fluorescent protein?
GFP: from the aequorea victorias. Emits green light (509nm) when excited with light (395nm).
Although now a whole spectrum of fluorescent proteins.
