Chapter 8: Methods in Cell Biology

Question 1

what are cell lines?

Answer 1

immortalized lines of donor cells crossed with cancer cells

Question 2

why are immortalized cell lines crossed with cancer cells?

Answer 2

encourages constant cell proliferation and prevents them from dying out like normal cells

Question 3

What is the BHK21 cell line?

Answer 3

a Syrian hamster fibroblast cell line

Question 4

What are the downsides of immortalized cell lines?

Answer 4

can only replicate a few times to avoid phenotypic drifting

Question 5

what are HELA cells?

Answer 5

highly robust human epithelial cancer cells that can easily contaminate other cultures

Question 6

how are hybridomas created? (3)

Answer 6

1. double nucleated heterokaryons created
2. cells are allowed to proliferate
3. hybrids are formed from nucleolytic crossing over

Question 7

what are heterokaryons?

Answer 7

double nucleated cells

Question 8

why are hybridomas used?

Answer 8

method for producing many pure cloned (myoclonal) antibodies; rather than polyclonal mixtures

Question 9

what speed would you spin to get whole cells, nuclei, and cytoskeleton?

Answer 9

low speed

Question 10

what speed would you spin to get mitochondria, lysosomes, and peroxisomes

Answer 10

mid speed

Question 11

what speed would you spin to get microsomes and vesicles?

Answer 11

high speed

Question 12

what speed would you spin to get ribosomes, viruses, and other large macromolecules?

Answer 12

very high speed

Question 13

this cell biology method passes sample through media by constantly applying solvent

Answer 13

column chromatography

Question 14

why is column chromatography used?

Answer 14

separates sample into fractions based on ions, affinity, size, etc.

Question 15

what are the steps for using epitope tags?

Answer 15

1. epitope tag is introduced to target gene
2. gene is introduced to cell
3. produces epitope-tagged protein
4. protein can then be used downstream

Question 16

what are two ways epitope-tagged proteins are used?

Answer 16

1. rapid purification from cell
2. visualized using fluorescence

Question 17

what is the problem with epitope-tagged proteins

Answer 17

tags can prevent proper folding; they can only be used for certain proteins (observation changes function)

Question 18

what is the purpose of SDS page electrophoresis?

Answer 18

determines molecular weight of whole proteins/subunits

Question 19

why is the substance SDS used in SDS page electrophoresis?

Answer 19

breaks disulfide bridges (encourages subunit formation) & effects charge

Question 20

this is a method of gel electrophoresis that separates samples based on isoelectric pH point

Answer 20

isoelectric focusing

Question 21

this is a combination of both SDS page and isoelectric focusing; creates two axes of visualization

Answer 21

2D electrophoresis

Question 22

what are some alterations that must be made to 2D electrophoresis to aid in visualization

Answer 22

1. need large amounts of protein
2. use silver-based dyes

Question 23

what are restriction enzymes typically used for?

Answer 23

generally used to customize bacterial plasmids

Question 24

what is the scientific purpose of using restriction endonuclease?

Answer 24

capable of recognizing and chopping up non-self DNA so that new sequences can be inserted

Question 25

What are some problems with using restriction endonucleases for gene editing?

Answer 25

1. it is a fixed behavior (not learned/memory)
2. in euks expression changes can ONLY work with correct promoters

Question 26

what is an example of the uses of plasmid cloning vectors?

Answer 26

insulin production

Question 27

how are plasmids cloned using vectors?

Answer 27

1. DNA fragment in ligated into the corresponding plasmid
2. plasmid inserted into bacteria
3. plasmids are cloned with cell proliferation
4. bacteria are lysed to release plasmid product

Question 28

what is the purpose of agarose electrophoresis?

Answer 28

used as a confirmation method of ideal product (i.e. correct plasmid isolation/product)

Question 29

How are samples separated in agarose gel?

Answer 29

by molecular weight (and charge if you try hard enough)

Question 30

What does FISH stand for?

Answer 30

Fluorescent in situ Hybridization

Question 31

what does in situ mean?

Answer 31

in place

Question 32

what is the FISH method used for?

Answer 32

mapping out the locations of target genes in mitotic chromosomes

Question 33

What are the 3 steps of PCR?

Answer 33

1. heating
2. annealing
3. elongation

Question 34

What happens when using expression vectors?

Answer 34

DNA sequence is inserted in a target before necessary promoter sequence

Question 35

why do we use expression vectors?

Answer 35

creates a temperature-dependent promotor sequence that can be controlled by adding heat

Question 36

What are the two origins to understanding how a protein/gene functions?

Answer 36

1. introduce altered gene to observe phenotypic change
2. biochemical tests of isolated protein to identify origin

Question 37

If you started with an unknown isolated protein, what tests would you do to find the corresponding gene?

Answer 37

1. X/NMR tests to identify domains and AAs
2. find corresponding codons/source mRNA
3. use target protein (dCas9) to find target location
4. then alter that region to see if protein changes

Question 38

what do microarrays do?

Answer 38

observe gene expression patterns at one specific slice of time

Question 39

what is the purpose of a false color image microarray?

Answer 39

observes gene expressions over time rather than just a single moment

Question 40

what are transgenic mice used for?

Answer 40

observing the results of altered embryos

Question 41

what is used to identify whether a plant is a GMO or not?

Answer 41

DNA barcode or functional genetic modifications

Question 42

what is the knockout substitute technique?

Answer 42

method of gene knockout through C. elegans and genetically altered E. coli

Question 43

What does RISC stand for?

Answer 43

RNA induced silencing complex