Chapter 8: Methods in Cell Biology Flashcards
what are cell lines?
immortalized lines of donor cells crossed with cancer cells
why are immortalized cell lines crossed with cancer cells?
encourages constant cell proliferation and prevents them from dying out like normal cells
What is the BHK21 cell line?
a Syrian hamster fibroblast cell line
What are the downsides of immortalized cell lines?
can only replicate a few times to avoid phenotypic drifting
what are HELA cells?
highly robust human epithelial cancer cells that can easily contaminate other cultures
how are hybridomas created? (3)
- double nucleated heterokaryons created
- cells are allowed to proliferate
- hybrids are formed from nucleolytic crossing over
what are heterokaryons?
double nucleated cells
why are hybridomas used?
method for producing many pure cloned (myoclonal) antibodies; rather than polyclonal mixtures
what speed would you spin to get whole cells, nuclei, and cytoskeleton?
low speed
what speed would you spin to get mitochondria, lysosomes, and peroxisomes
mid speed
what speed would you spin to get microsomes and vesicles?
high speed
what speed would you spin to get ribosomes, viruses, and other large macromolecules?
very high speed
this cell biology method passes sample through media by constantly applying solvent
column chromatography
why is column chromatography used?
separates sample into fractions based on ions, affinity, size, etc.
what are the steps for using epitope tags?
- epitope tag is introduced to target gene
- gene is introduced to cell
- produces epitope-tagged protein
- protein can then be used downstream
what are two ways epitope-tagged proteins are used?
- rapid purification from cell
- visualized using fluorescence
what is the problem with epitope-tagged proteins
tags can prevent proper folding; they can only be used for certain proteins (observation changes function)
what is the purpose of SDS page electrophoresis?
determines molecular weight of whole proteins/subunits
why is the substance SDS used in SDS page electrophoresis?
breaks disulfide bridges (encourages subunit formation) & effects charge
this is a method of gel electrophoresis that separates samples based on isoelectric pH point
isoelectric focusing
this is a combination of both SDS page and isoelectric focusing; creates two axes of visualization
2D electrophoresis
what are some alterations that must be made to 2D electrophoresis to aid in visualization
- need large amounts of protein
- use silver-based dyes
what are restriction enzymes typically used for?
generally used to customize bacterial plasmids
what is the scientific purpose of using restriction endonuclease?
capable of recognizing and chopping up non-self DNA so that new sequences can be inserted
What are some problems with using restriction endonucleases for gene editing?
- it is a fixed behavior (not learned/memory)
- in euks expression changes can ONLY work with correct promoters
what is an example of the uses of plasmid cloning vectors?
insulin production
how are plasmids cloned using vectors?
- DNA fragment in ligated into the corresponding plasmid
- plasmid inserted into bacteria
- plasmids are cloned with cell proliferation
- bacteria are lysed to release plasmid product
what is the purpose of agarose electrophoresis?
used as a confirmation method of ideal product (i.e. correct plasmid isolation/product)
How are samples separated in agarose gel?
by molecular weight (and charge if you try hard enough)
What does FISH stand for?
Fluorescent in situ Hybridization
what does in situ mean?
in place
what is the FISH method used for?
mapping out the locations of target genes in mitotic chromosomes
What are the 3 steps of PCR?
- heating
- annealing
- elongation
What happens when using expression vectors?
DNA sequence is inserted in a target before necessary promoter sequence
why do we use expression vectors?
creates a temperature-dependent promotor sequence that can be controlled by adding heat
What are the two origins to understanding how a protein/gene functions?
- introduce altered gene to observe phenotypic change
- biochemical tests of isolated protein to identify origin
If you started with an unknown isolated protein, what tests would you do to find the corresponding gene?
- X/NMR tests to identify domains and AAs
- find corresponding codons/source mRNA
- use target protein (dCas9) to find target location
- then alter that region to see if protein changes
what do microarrays do?
observe gene expression patterns at one specific slice of time
what is the purpose of a false color image microarray?
observes gene expressions over time rather than just a single moment
what are transgenic mice used for?
observing the results of altered embryos
what is used to identify whether a plant is a GMO or not?
DNA barcode or functional genetic modifications
what is the knockout substitute technique?
method of gene knockout through C. elegans and genetically altered E. coli
What does RISC stand for?
RNA induced silencing complex
