Cell Bio Ch 8 Flashcards
FACS is?
Florescence activated cell sorter
Uses an antibody coupled to fluorescent dye, the antibody binds to only 1 cell type.
Single fluorescent cell gets a negative charge, and the non fluorescent get a positive charge. Accurate to 1 in a thousand cells.
Cells isolated from intact tissue
Extracellular membrane is disrupted by proteolytic enzymes.
EDTA chelates calcium on which the cell-cell adhesions depend.
Microdissection technique
Selected cells are dissected from thin tissue slices that are prepared for microscopic examination
Cut with laser
Disadvantages of using intact tissue for experiments
Not a convenient source of material.
Difficult to purify particular molecules from an intact tissue (too complex).
No way possible to do genetic manipulations on the livestock.
Cultured cells ____________
Continue to differentiate similar to their origin cells
Advantages of using cells from cultures for experiments
It provides a more homogeneous population of cells to extract material.
Convenient to work with in the lab.
Under proper conditions, most plants and animal cells can live, multiply and even differentiate in a tissue-culture dish.
In the lab they can be monitored continuously and effect of different media or hormones can be studied.
In addition, interactions between two cell types can also be studied.
Experiments done on cultured cells are called “in vitro” (“in glass”) in contrast to those done using intact organism “in vivo” (“ in the living organism”).
Primary culture
Culture prepared directly from tissue of the organism.
Secondary culture
Primary culture has been repeatedly cultured
____________ cells are widely used as a source of homogenous cells.
Eukaryotic
Problem with cell cultures
Cells die after finite number of cell divisions
replicative cell senescence
Cell death after a finite number of cell divisions.
2 main reason for cell death
Telomere shortening - cells have turned off production of Telomerase enzyme. As a result telomere shortening occurs with each cell division.
Activation of cell –cycle checkpoint mechanism that arrests cell cycle and is called “culture shock”.
How to overcome cell death
Provide telomerase to the cells.
Add oncogene to inactivate the checkpoint.
Most human cells used in labs are cancer cells
Transformed cell lines
Human cells used in lab, derived from cancer cells
Rodent cell lines
Do not turn off telomerase production. Can grow indefinitely if culture shock is avoided.
Embryonic stem cells are harvested from
Inner mass of early embryo
Advantages of embryonic stem cells
Can provide specialized cell therapy:
replace degenerate muscles in muscular dystrophy.
replace insulin secreting cells (Diabetes type I).
replace dead nerves in Parkinson’s disease.
rejuvenate cardiac muscles in weak heart cells.
Disadvantages of embryonic stem cells
If the transplanted cells are genetically different from the patient’s cells it could be rejected by the patient’s immune system. This could be avoided by using cells derived from the patient.
If the ES cells are themselves transplanted into a person they can give rise to tumors called teratomas.
a
Cells from adult tissue can be used for
Reproductive cloning-creating another organism or tissue through cloning
Or therapeutic cloning- creating generalized personalized embryonic stem cells
Therapeutic cloning
Identical to donor cells
Can be used for tissue repair
Can be used to study inherited disease
Column chromatography
Used to separate proteins
Mixture of protein passed through a solid porous matrix
Different proteins are retarded at different rates by their interaction with the matrix
High degree of purity can be obtained
Different matrices of chromatography
Ion exchange chromatography- proteins separated on basis of surface charge using positive or negative charged beads
Hydrophobic chromatography- binds proteins with exposed hydrophobic regions
Gel-filtration chromatography- separates proteins by their size
Affinity chromatography- proteins separated by their binding properties
Epitope tagging
To see the localization or purification of proteins
Small tag (epitope) targets protein using an antibody
Attaches to N-terminus or C-terminus
Commonly used epitope tags for purification
Flag, GST, 6xHis, c-myc
SDS Polyacrylamide-Gel Electrophoresis (SDS-PAGE)
Highly cross linked gel
Pore size of gel can be adjusted to retard protein of interest
Proteins are in a negatively charged substance sodium dodecyl sulfate, SDS
SDS Binds to hydrophobic regions, unfolding proteins a,d separating proteins from other proteins and lipids
Separated by molecular weight. SDS allows proteins to be pulled toward anode
Dye Coomassie blue, silver stain, to allow detection
Yeast two-hybrid system
DNA gene activator protein and bind domain need to be close in proximity to activate transcription
Bait and prey proteins attached, once attracted to each other, transcription is initiated.
Genetic engineering
The ability to manipulate DNA with precision in a test tube or an organism.
Recombinant DNA technology
A mixture of techniques that are used to manipulate DNA
Restriction enzymes
Take large DNA molecules and cut them in fragments.
Bacteria use them to cut up foreign DNA that meters the cell
Deoxyribo endonucleases
Cut palindromic sequences
Restriction enzyme uses
No ATP required
Can make sticky or blunt ends
Can recognize 4, 6, or more bases for cleavage
6 base is called a “six-base cutter”
Gel electrophoresis with DNA
DNA is negatively charged and doesn’t need SDS, like proteins do.
Ethidium bromide used for dye
Porous agarose used for gel
PCR
Genes can be selectively amplified, usually 20-30 cycles
1 cycle, 3 steps:
Denaturation- at 95C to separate DNA strands
Annealing- at 55C to allow primers to hybridize
Elongation- at 68C so polymerase can elongate the chain
