Chapter 9 Flashcards
Visualizing cells
What is GFP?
Green fluorescent protein
The gene encoding GFP is inserted into a plasmid before or after a gene of interest and is then expressed in cells
Fluorescent protein that is widely used as a marker for monitoring the movement of proteins in living cells
What is the limit of resolution?
The minimal separation between two objects at which they appear distinct
What is Bright Field microscopy?
The normal light miscopy method in which the image is obtained by simple transmission of light through the object being viewed
What is fluorescence microscopy?
Similar to light microscopy but the illumination light is passes through one set of filters before the specimen, to select those wavelengths that excite the dye, and through another set of filters before it reaches the eye, to select only those wavelengths emitted when the dye fluoresces
What is confocal microscopy?
Type of light microscope that produces a clear image of a given plane within a solid object. It uses a laser beam as a pinpoint source of illumination and scans across the plane to produce a two-dimensional optical section
What is FRET?
Fluorescence resonance energy transfer
What is FRAP?
Fluorescence recovery after photobleaching?
As which nm does DNA bases maximally absorb at? Will ultraviolet light damage the DNA when using dye on living cells?
260 nm
UV light will potentially damage DNA. But by confining the illuminating beam to wavelength well above 260 nm or using filters to block the damaging wavelenght, damage can be avoided
What are the advantages and disadvantages of secondary antibodies that carry fluorescent tags vs. bound enzymes?
Both amplify initial signal
- Fluorescent tags amplify several-fold
- Bound enzymes amplify more than 1000-fold
Enzymes-linked methods are very sensitive, but diffusion of the reaction product away from the enzyme limits the spatial resolution
How can a plasmid be transferred into a cell?
Can be introduced into a cell by lipofection
How can you establish a cell line that stably expresses a GFP-tagged protein?
To establish a stable expressing cell line, the DNA will be introduced into the cells genome.
A marker gene i co-transfected into the cell, so both the gene of interest and the marker gene is in the plasmid and introduced into the cell
What are the advantages of using GFP instead of antibodies targeting a protein?
With GFP it is possible to study the cell cycle with live cell imaging.
Immunostaining with antibodies kills the cell
What is the use of CRISPR/Cas9?
Can be used to make DNA double strand breaks at unique sites in the genome
By then inserting a gene encoding GFP next to a gene encoding a signalling protein of interest, the now stable expression of GFP tagged signalling protein can be observed with live-cell imaging
Can also be used to localize a gene of interest by fusing Cas9 with gene encoding GFP. A guide RNA can then target Cas9-GFP to a specific gene loci
What is the purpose of rescue experiments?
The purpose of a rescue experiment is to see is the genes are introduced again, the phenotype of the cell will go back to normal, and the observed phenotype is due to the knock-down or knock-out of the gene and not due to the treatment of the cell.
What is differential-interference-contrast microscopy?
Used to enhance the contrast in unstained, transparent samples
Complex optical system produces an image with the object appearing black to white on a grey background.
