Lecture 12

Question 1

why is authentication important

Answer 1

if cells are not what they are supp[osed to be, the results are at best compromised, and at worst invalid

Question 2

explain the conundrum of cells with short doubling times and high plating efficiency

Answer 2

they are very valuable for experiments because they grow so well, but they are also a hazard because they easily cross contaminate other cell lines. lots of “victim cell lines” result from misidentified cells that a highly efficient line invaded (HeLa

Question 3

common causes of misidentification and cross-contamination

Answer 3

importing non-authenticated stock
over enthusiasm by originator
poor technique: unplugged pipettes, poor observation, not counting, handling >1 line at a time, sharing medium, mislabeling

Question 4

describe methods to verify authenticity and identify individuals

Answer 4

DNA fingerprint: use restriction enzyme digest and probe. compare unique banding patterns
DNA STR profiling: short tandem repeats are polymorphic markers in genome, unique to individual. 80% match confirms authenticity

Question 5

describe methods to verify authenticity and identify species

Answer 5

DNA barcoding: use “barcode region” in cytochrome oxidase gene I region, unique to species. produce small DNA fragments, sequence, and compare
Isozyme electrophoresis: species vary in size of certain enzymes, use target proteins that are conserved but variable. useful for interspecies cell contamination
Chromosome content/banding: count and look at morphology of chromosomes, create karyotype. Karyotype allows species, sex, and normal vs transformed identification. Best define method!!

Question 6

chromosome numbers of human, rat, and mouse

Answer 6

human: 46
mouse: 40
rat: 42

Question 7

how are chromosomal assays prepared

Answer 7

1. block metaphase with colchicine
2. collect by trypsinization or shake off
3. sell with hypotonic treatment
4. fix and wash
5. spread on slides
6. stain (giemsa, hoechst33258)
   can get banding by pretreating with trypsin (to digest some chromosome sections) and special stains
7. analysis

Question 8

chromosome banding techniques

Answer 8

quinacrine banding (Q banding) uses fluorescent intercalating staining and binds stronger to the less dense areas.
giemsa banding (G banding) uses trypsin to digest less dense regions and stains darker on more dense areas
painting: additional resolution and specificity, each chromosome is a unique color. limited species
SKY and M-FISH. diff colors for each, more sensitive, identifies chromosomal alterations (recombination)

Question 9

define requirements for characterization

Answer 9

1. authentication
2. confirmation of species of origin
3. correlation with tissue of origin
4. transformation status
5. indication if prone to genetic instability
6. any phenotypic variation
7. specific cell line identification

Question 10

how is cell morphology used in characterization?

Answer 10

simplest and most direct method, has significant shortcomings. changes in different conditions, hard to distinguish between cell types.
comparative observations are key: compare at same growth stage, density, medium, and substrate. apples to apples!

Question 11

how are lineage markers used in characterization? examples of markers for stem cells, cell surface antigens, and intermediate filaments

Answer 11

cells acquire lineage markers as they differentiate. the markers indicate relationship of a particular cell line to its tissue of origin. markers can be identified by gene transcription (RTPCR), protein presence (western), or functional assay for differentiation
stem cell markers: SSEA-1 (surface), OCT4, NANOG
cell surface antigens: HLA, useful in cell sorting
Intermediate filament proteins: glial fibrillary acid protein (astrocytes), desmin (muscles), cytokeratin (epithelial cells)