Biotech Final - Lecture 6 Flashcards
What is the difference between primary cell lines and continuous cell lines?
Primary cell lines are those cultured directly from a subject. These present a limited lifespan.
Continuous or immortal cell lines have acquired the ability to proliferate infinitely. (Cancer cell line or addition of the telomerase gene)
What is the difference between adherent cell lines and suspension cell lines?
Adherent cells require a surface (anchorage dependence), form a monolayer.
Some cells naturally live in suspension, such as cells that exist in the bloodstream. There also exists cells that can be modified to be able to survive in suspension.
What are the different applications of cell culture?
1) Investigate cell physiology and/or biological function of a single gene product
2) Test the effect of compounds on specific cell types or cellular situations
3) Synthesize biologicals
4) Formation of monoclonal antibodies
What are Hek 293 cells?
Human embryonic kidney cells.
What are cell culture techniques?
- Splitting pre-initiated cells.
- Quantification of cell numbers.
- Subculture onto plates for transfection and form a stock plate
What are overexpression techniques?
- Transfection
2. Cell lysis
Describe the steps of lipoplex mediated transfection and endocytosis.
1) DNA forms complex with liposome
2) This is taken in by non-specific endocytosis
3) Endosome formation
4) Either endosomal maturation or ensome with lipid mixing
4. a) endosomal maturation leads to DNA fragmentation and lysosome degradation
4. b) Endosome with lipid mixing leads to escape form the endosome and entry into nucleus for expression.
What are the steps of detergent cell lysis?
Start with plate containing transfected cells.
1) Remove media
2) Wash with PBS
3) Lyse with detergent containing buffer (Protease inhibitors)
4) Centrifuge and keep supernatant
5) Freeze
