Cell Bio Ch 8

Question 0

FACS is?

Answer 0

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.

Question 1

Cells isolated from intact tissue

Answer 1

Extracellular membrane is disrupted by proteolytic enzymes.

EDTA chelates calcium on which the cell-cell adhesions depend.

Question 2

Microdissection technique

Answer 2

Selected cells are dissected from thin tissue slices that are prepared for microscopic examination
Cut with laser

Question 3

Disadvantages of using intact tissue for experiments

Answer 3

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.

Question 4

Cultured cells ____________

Answer 4

Continue to differentiate similar to their origin cells

Question 5

Advantages of using cells from cultures for experiments

Answer 5

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”).

Question 6

Primary culture

Answer 6

Culture prepared directly from tissue of the organism.

Question 7

Secondary culture

Answer 7

Primary culture has been repeatedly cultured

Question 8

____________ cells are widely used as a source of homogenous cells.

Answer 8

Eukaryotic

Question 9

Problem with cell cultures

Answer 9

Cells die after finite number of cell divisions

Question 10

replicative cell senescence

Answer 10

Cell death after a finite number of cell divisions.

Question 11

2 main reason for cell death

Answer 11

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”.

Question 12

How to overcome cell death

Answer 12

Provide telomerase to the cells.
Add oncogene to inactivate the checkpoint.

Most human cells used in labs are cancer cells

Question 13

Transformed cell lines

Answer 13

Human cells used in lab, derived from cancer cells

Question 14

Rodent cell lines

Answer 14

Do not turn off telomerase production. Can grow indefinitely if culture shock is avoided.

Question 15

Embryonic stem cells are harvested from

Answer 15

Inner mass of early embryo

Question 16

Advantages of embryonic stem cells

Answer 16

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.

Question 17

Disadvantages of embryonic stem cells

Answer 17

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

Question 18

Cells from adult tissue can be used for

Answer 18

Reproductive cloning-creating another organism or tissue through cloning
Or therapeutic cloning- creating generalized personalized embryonic stem cells

Question 19

Therapeutic cloning

Answer 19

Identical to donor cells
Can be used for tissue repair
Can be used to study inherited disease

Question 20

Column chromatography

Answer 20

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

Question 21

Different matrices of chromatography

Answer 21

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

Question 22

Epitope tagging

Answer 22

To see the localization or purification of proteins
Small tag (epitope) targets protein using an antibody
Attaches to N-terminus or C-terminus

Question 23

Commonly used epitope tags for purification

Answer 23

Flag, GST, 6xHis, c-myc

Question 24

SDS Polyacrylamide-Gel Electrophoresis (SDS-PAGE)

Answer 24

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

Question 25

Yeast two-hybrid system

Answer 25

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.

Question 26

Genetic engineering

Answer 26

The ability to manipulate DNA with precision in a test tube or an organism.

Question 27

Recombinant DNA technology

Answer 27

A mixture of techniques that are used to manipulate DNA

Question 28

Restriction enzymes

Answer 28

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

Question 29

Restriction enzyme uses

Answer 29

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"

Question 30

Gel electrophoresis with DNA

Answer 30

DNA is negatively charged and doesn't need SDS, like proteins do. Ethidium bromide used for dye Porous agarose used for gel

Question 31

PCR

Answer 31

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